Diagnostic method based on quantification of extramitochondrial DNA

ABSTRACT

Compositions and methods based on quantification of extramitochondrial DNA (exmtDNA) sequences are provided that are useful for detecting the presence of or risk for having a disease associated with altered mitochondrial function, and for identifying agents suitable for treating such diseases. The exmtDNA sequences have strong homology to authentic mitochondrial DNA (mtDNA) sequences.

TECHNICAL FIELD

The present invention relates generally to diseases in which altered mitochondrial function, such as free radical mediated oxidative injury, leads to tissue degeneration and, more specifically, to compositions and methods for detecting predisposition to such diseases by quantifying extramitochondrial DNA.

BACKGROUND OF THE INVENTION

A number of degenerative diseases are thought to be caused by or be associated with alterations in mitochondrial function. These diseases include Alzheimer's Disease, diabetes mellitus, Parkinson's Disease, Huntington's disease, dystonia, Leber's hereditary optic neuropathy, schizophrenia, and myodegenerative disorders such as “mitochondrial encephalopathy, lactic acidosis, and stroke” (MELAS), and “myoclonic epilepsy ragged red fiber syndrome” (MERRF). Other diseases involving altered metabolism or respiration within cells may also be regarded as diseases associated with altered mitochondrial function.

Functional mitochondria contain gene products encoded by mitochondrial genes situated in mitochondrial DNA (mtDNA) and by extramitochondrial genes not situated in the circular mitochondrial genome. The 16.5 kb mtDNA encodes 22 tRNAs, two ribosomal RNAs (rRNA) and only 13 enzymes of the electron transport chain (ETC), the elaborate multi-complex mitochondrial assembly where, for example, respiratory oxidative phosphorylation takes place. The overwhelming majority of mitochondrial structural and functional proteins are encoded by extramitochondrial, and in most cases presumably nuclear, genes. Accordingly, mitochondrial and extramitochondrial genes may interact directly, or indirectly via gene products and their downstream intermediates, including metabolites, catabolites, substrates, precursors, cofactors and the like. Alterations in mitochondrial function, for example impaired electron transport activity, defective oxidative phosphorylation or increased free radical production, may therefore arise as the result of defective mtDNA, defective extramitochondrial DNA, defective mitochondrial or extramitochondrial gene products, defective downstream intermediates or a combination of these and other factors.

Mitochondria are the subcellular organelles that manufacture bioenergetically essential adenosine triphosphate (ATP) by oxidative phosphorylation. Defective mitochondrial activity, including failure at any step of the ETC, may result in the generation of highly reactive free radicals that have the potential of damaging cells and tissues. These free radicals may include reactive oxygen species (ROS) such as superoxide, peroxynitrite and hydroxyl radicals, and potentially other reactive species that may be toxic to cells. For example, oxygen free radical induced lipid peroxidation is a well established pathogenetic mechanism in central nervous system (CNS) injury, such as that found in a number of degenerative diseases, and in ischemia (i.e., stroke).

There are at least two deleterious consequences of exposure to reactive free radicals arising from mitochondrial dysfunction that adversely impact the mitochondria themselves. First, free radical mediated damage may inactivate one or more of the myriad proteins of the ETC. According to generally accepted theories of mitochondrial function, proper ETC respiratory activity requires maintenance of an electrochemical potential in the inner mitochondrial membrane by a coupled chemiosmotic mechanism. Free radical oxidative activity may dissipate this membrane potential, thereby preventing ATP biosynthesis and halting the production of a vital biochemical energy source. In addition, mitochondrial proteins such as cytochrome c and “apoptosis inducing factor” may leak out of the mitochondria after permeability transition and may induce the genetically programmed cell suicide sequence known as apoptosis or programmed cell death (PCD).

Second, free radical mediated damage may result in catastrophic mitochondrial collapse that has been termed “transition permeability”. For example, rapid mitochondrial permeability transition likely entails changes in the inner mitochondrial transmembrane protein adenylate translocase that results in the formation of a “pore.” In any event, because permeability transition is potentiated by free radical exposure, it may be more likely to occur in the mitochondria of cells from patients having mitochondria associated diseases that are chronically exposed to such reactive free radicals.

Altered mitochondrial function characteristic of the mitochondria associated diseases may also be related to loss of mitochondrial membrane electrochemical potential by mechanisms other than free radical oxidation, and such transition permeability may result from direct or indirect effects of mitochondrial genes, gene products or related downstream mediator molecules and/or extramitochondrial genes, gene products or related downstream mediators, or from other known or unknown causes.

Diabetes mellitus is a common, degenerative disease affecting 5 to 10 percent of the population in developed countries. The propensity for developing diabetes mellitus is reportedly maternally inherited, suggesting a mitochondrial genetic involvement. (Alcolado, J. C. and Alcolado, R., Br. Med. J. 302:1178-1180 (1991); Reny, S. L., International J. Epidem. 23:886-890 (1994)). Diabetes is a heterogenous disorder with a strong genetic component; monozygotic twins are highly concordant and there is a high incidence of the disease among first degree relatives of affected individuals.

At the cellular level, the degenerative phenotype that may be characteristic of late onset diabetes mellitus includes indicators of altered mitochondrial respiratory function, for example impaired insulin secretion, decreased ATP synthesis and increased levels of reactive oxygen species. Studies have shown that diabetes mellitus may be preceded by or associated with certain related disorders. For example, it is estimated that forty million individuals in the U.S. suffer from late onset impaired glucose tolerance (IGT). IGT patients fail to respond to glucose with increased insulin secretion. A small percentage of IGT individuals (5-10%) progress to insulin deficient non-insulin dependent diabetes (NIDDM) each year. Some of these individuals further progress to insulin dependent diabetes mellitus (IDDM). These forms of diabetes mellitus, NIDDM and IDDM, are associated with decreased release of insulin by pancreatic beta cells and/or a decreased end-organ response to insulin. Other symptoms of diabetes mellitus and conditions that precede or are associated with diabetes mellitus include obesity, vascular pathologies, peripheral and sensory neuropathies, blindness and deafness.

Parkinson's disease (PD) is a progressive, neurodegenerative disorder associated with altered mitochondrial function and characterized by the loss and/or atrophy of dopamine-containing neurons in the pars compacta of the substantia nigra of the brain. Like Alzheimer's Disease (AD), PD also afflicts the elderly. It is characterized by bradykinesia (slow movement), rigidity and a resting tremor. Although L-Dopa treatment reduces tremors in most patients for a while, ultimately the tremors become more and more uncontrollable, making it difficult or impossible for patients to even feed themselves or meet their own basic hygiene needs.

It has been shown that the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) induces parkinsonism in animals and man at least in part through its effects on mitochondria. MPTP is converted to its active metabolite, MPP+, in dopamine neurons; it then becomes concentrated in the mitochondria. The MPP+ then selectively inhibits the mitochondrial enzyme NADH:ubiquinone oxidoreductase (“Complex I”), leading to the increased production of free radicals, reduced production of adenosine triphosphate, and ultimately, the death of affected dopamine neurons.

Mitochondrial Complex I is composed of 40-50 subunits; most are encoded by the nuclear genome and seven by the mitochondrial genome. Since parkinsonism may be induced by exposure to mitochondrial toxins that affect Complex I activity, it appears likely that defects in Complex I proteins may contribute to the pathogenesis of PD by causing a similar biochemical deficiency in Complex I activity. Indeed, defects in mitochondrial Complex I activity have been reported in the blood and brain of PD patients (Parker et al., Am. J. Neurol. 26:719-723, 1989).

Alzheimer's disease (AD) is a progressive neurodegenerative disorder that is characterized by loss and/or atrophy of neurons in discrete regions of the brain, and that is accompanied by extracellular deposits of β-amyloid and the intracellular accumulation of neurofibrillary tangles. It is a uniquely human disease, affecting over 13 million people worldwide. It is also a uniquely tragic disease. Many individuals who have lived normal, productive lives are slowly stricken with AD as they grow older, and the disease gradually robs them of their memory and other mental faculties. Eventually, they cease to recognize family and loved ones, and they often require continuous care until their eventual death.

There is evidence that defects in oxidative phosphorylation within the mitochondria are at least a partial cause of sporadic AD. The enzyme cytochrome c oxidase (COX), which makes up part of the mitochondrial electron transport chain (ETC), is present in normal amounts in AD patients; however, the catalytic activity of this enzyme in AD patients and in the brains of AD patients at autopsy has been found to be abnormally low. This suggests that the COX in AD patients is defective, leading to decreased catalytic activity that in some fashion causes or contributes to the symptoms that are characteristic of AD.

Focal defects in energy metabolism in the mitochondria, with accompanying increases in oxidative stress, may be associated with AD. It is well-established that energy metabolism is impaired in AD brain (Palmer et al., Brain Res. 645:338-42, 1994; Pappolla et al., Am. J. Pathol. 140:621-28, 1992; Jeandel et al., Gerontol. 35:275, 1989; Balazs et al., Neurochem. Res. 19:1131-37, 1994; Mecocci et al., Ann. Neurol. 36:747-751, 1994; Gsell et al., J. Neurochem. 64:1216-23, 1995). For example, regionally specific deficits in energy metabolism in AD brains have been reported in a number of positron emission tomography studies (Kuhl, et al., J. Cereb. Blood Flow Metab. 7:S406, 1987; Grady, et al., J. Clin. Exp. Neuropsychol. 10:576-96, 1988; Haxby et al., Arch. Neurol. 47:753-60, 1990; Azari et al., J. Cereb. Blood Flow Metab. 13:438-47, 1993). Metabolic defects in the temporoparietal neocortex of AD patients apparently presage cognitive decline by several years. Skin fibroblasts from AD patients display decreased glucose utilization and increased oxidation of glucose, leading to the formation of glycosylation end products (Yan et al., Proc. Nat. Acad. Sci. USA 91:7787-91, 1994). Cortical tissue from postmortem AD brain shows decreased activity of the mitochondrial enzymes pyruvate dehydrogenase (Sheu et al., Ann. Neurol. 17:444-49, 1985) and α-ketoglutarate dehydrogenase (Mastrogiacomo et al., J. Neurochem. 6:2007-14, 1994), which are both key enzymes in energy metabolism. Functional magnetic resonance spectroscopy studies have shown increased levels of inorganic phosphate relative to phosphocreatine in AD brain, suggesting an accumulation of precursors that arises from decreased ATP production by mitochondria (Pettegrew et al., Neurobiol. of Aging 15:117-32, 1994; Pettigrew et al., Neurobiol. of Aging 16:973-75, 1995). In addition, the levels of pyruvate, but not of glucose or lactate, are reported to be increased in the cerebrospinal fluid of AD patients, consistent with defects in cerebral mitochondrial electron transport chain (ETC) activity (Parnetti et al., Neurosci. Lett. 199:231-33, 1995).

Signs of oxidative injury are prominent features of AD pathology and, as noted above, reactive oxygen species (ROS) are critical mediators of neuronal degeneration. Indeed, studies at autopsy show that markers of protein, DNA and lipid peroxidation are increased in AD brain (Palmer et al., Brain Res. 645:338-42, 1994; Pappolla et al., Am. J. Pathol. 140:621-28, 1992; Jeandel et al., Gerontol. 35:275-82, 1989; Balazs et al., Arch. Neurol. 4:864, 1994; Mecocci et al., Ann. Neurol. 36:747-51, 1994; Smith et al., Proc. Nat. Acad. Sci. USA 88:10540-43, 1991). In hippocampal tissue from AD but not from controls, carbonyl formation indicative of protein oxidation is increased in neuronal cytoplasm, and nuclei of neurons and glia (Smith et al., Nature 382:120-21, 1996). Neurofibrillary tangles also appear to be prominent sites of protein oxidation (Schweers et al., Proc. Nat. Acad. Sci. USA 92:8463, 1995; Blass et al., Arch. Neurol. 4:864, 1990). Under stressed and non-stressed conditions incubation of cortical tissue from AD brains taken at autopsy demonstrate increased free radical production relative to non-AD controls. In addition, the activities of critical antioxidant enzymes, particularly catalase, are reduced in AD (Gsell et al., J. Neurochem. 64:1216-23, 1995), suggesting that the AD brain is vulnerable to increased ROS production. Thus, oxidative stress may contribute significantly to the pathology of mitochondria associated diseases such as AD, where mitochondrial dysfunction and/or elevated ROS may be present.

One hallmark pathology of AD is the death of selected neuronal populations in discrete regions of the brain. Cell death in AD is presumed to be apoptotic because signs of programmed cell death (PCD) are seen and indicators of active gliosis and necrosis are not found. (Smale et al., Exp. Neurolog. 133:225-230, 1995; Cotman et al., Molec. Neurobiol. 10:19-45, 1995.) The consequences of cell death in AD, neuronal and synaptic loss, are closely associated with the clinical diagnosis of AD and are highly correlated with the degree of dementia in AD (DeKosky et al., Ann. Neurology 27:457-464, 1990).

Mitochondrial dysfunction is thought to be critical in the cascade of events leading to apoptosis in various cell types (Kroemer et al., FASEB J. 9:1277-87, 1995), and may be a cause of apoptotic cell death in neurons of the AD brain. Altered mitochondrial physiology may be among the earliest events in PCD (Zamzami et al., J. Exp. Med. 182:367-77, 1995; Zamzami et al., J. Exp. Med. 181:1661-72, 1995) and elevated reactive oxygen species (ROS) levels that result from such altered mitochondrial function may initiate the apoptotic cascade (Ausserer et al., Mol. Cell. Biol. 14:5032-42, 1994). In several cell types, including neurons, reduction in the mitochondrial membrane potential (ΔΨm) precedes the nuclear DNA degradation that accompanies apoptosis. In cell-free systems, mitochondrial, but not nuclear, enriched fractions are capable of inducing nuclear apoptosis (Newmeyer et al., Cell 70:353-64, 1994). Perturbation of mitochondrial respiratory activity leading to altered cellular metabolic states, such as elevated intracellular ROS, may occur in mitochondria associated diseases and may further induce pathogenetic events via apoptotic mechanisms.

Oxidatively stressed mitochondria may release a preformed soluble factor that can induce chromosomal condensation, an event preceding apoptosis (Marchetti et al., Cancer Res. 56:2033-38, 1996). In addition, members of the Bcl-2 family of anti-apoptosis gene products are located within the outer mitochondrial membrane (Monaghan et al., J. Histochem. Cytochem. 40:1819-25, 1992) and these proteins appear to protect membranes from oxidative stress (Korsmeyer et al, Biochim. Biophys. Act. 1271:63, 1995). Localization of Bcl-2 to this membrane appears to be indispensable for modulation of apoptosis (Nguyen et al., J. Biol. Chem. 269:16521-24, 1994). Thus, changes in mitochondrial physiology may be important mediators of apoptosis. To the extent that apoptotic cell death is a prominent feature of neuronal loss in AD, mitochondrial dysfunction may be critical to the progression of this disease and may also be a contributing factor in other mitochondria associated diseases.

Regardless of whether a defect underlying a disease associated with altered mitochondrial function may have mitochondrial or extramitochondrial origins, and regardless of whether a defect underlying altered mitochondrial function has been identified, the present invention provides methods that are useful for determining the risk or presence of diseases associated with such altered mitochondrial function, and for identifying agents that are suitable for treating such diseases. In particular, as is elaborated herein below, the present invention provides compositions and methods for the detection of diseases associated with altered mitochondrial function by quantification of unusual mtDNA-like sequences not found in mitochondria and referred to as extramitochondrial DNA (exmtDNA), and other related advantages.

SUMMARY OF THE INVENTION

Briefly stated, the present invention is directed to compositions and methods useful for detecting mitochondria associated diseases and involving extramitochondrial DNA (exmtDNA) sequences that are highly homologous to mitochondrial DNA (mtDNA). In one aspect the invention provides a method for determining the risk for or presence of a disease associated with altered mitochondrial function in a first subject suspected of having or being at risk for having such a disease, by comparing a ratio r for each of a first and a second biological sample containing extramitochondrial DNA and mitochondrial DNA, the first biological sample being obtained from the first subject and the second sample being obtained from a second subject known to be free of a risk or presence of a disease associated with altered mitochondrial function, using the formula:

r=x/(x+y)

wherein x is the amount of extramitochondrial DNA in a sample, and y is the amount of mitochondrial DNA in the sample; and therefrom determining the risk or presence of the disease. In an embodiment of the invention, the ratio r is calculated by a method that comprises contacting a biological sample containing extramitochondrial DNA and mitochondrial DNA with an oligonucleotide primer having a nucleotide sequence that is complementary to a sequence present in the extramitochondrial DNA and present in the mitochondrial DNA, under conditions and for a time sufficient to allow hybridization of the primer to the extramitochondrial DNA and to the mitochondrial DNA; and detecting hybridization of the primer to the extramitochondrial DNA and to the mitochondrial DNA, in order to therefrom quantify the extramitochondrial DNA and the mitochondrial DNA.

In another embodiment, the ratio r is calculated by a method comprising contacting a sample containing amplified extramitochondrial DNA and mitochondrial DNA with an oligonucleotide primer having a nucleotide sequence that is complementary to a sequence present in the amplified extramitochondrial DNA and present in the amplified mitochondrial DNA, under conditions and for a time sufficient to allow hybridization of the primer to the extramitochondrial DNA and to the mitochondrial DNA; and detecting hybridization of the primer to the extramitochondrial DNA and to the mitochondrial DNA, and therefrom quantifying the extramitochondrial DNA and the mitochondrial DNA. In another embodiment of this aspect of the invention the ratio r is calculated by a method comprising contacting a biological sample containing extramitochondrial DNA and mitochondrial DNA with an oligonucleotide primer having a nucleotide sequence that is complementary to a sequence present in the extramitochondrial DNA and present in the mitochondrial DNA, under conditions and for a time sufficient to allow hybridization of said primer to the extramitochondrial DNA and to the mitochondrial DNA; and detecting hybridization and extension of the primer to the extramitochondrial DNA to produce a first product and hybridization and extension of the primer to the mitochondrial DNA to produce a second product distinguishable from the first product, and therefrom quantifying the extramitochondrial DNA and the mitochondrial DNA.

In another embodiment of this aspect of the invention the ratio r is calculated by a method comprising contacting a sample containing amplified extramitochondrial DNA and mitochondrial DNA with an oligonucleotide primer having a nucleotide sequence that is complementary to a sequence present in the amplified extramitochondrial DNA and present in the amplified mitochondrial DNA, under conditions and for a time sufficient to allow hybridization of the primer to the extramitochondrial DNA and to the mitochondrial DNA; and detecting hybridization and extension of the primer to the extramitochondrial DNA to produce a first product and hybridization and extension of the primer to the mitochondrial DNA to produce a second product distinguishable from the first product, and therefrom quantifying the extramitochondrial DNA and the mitochondrial DNA.

In another embodiment of this aspect of the invention the biological sample is treated by heating it in water to lyse cells contained in the sample, and then extracting cellular DNA from the lysed cells using an aqueous DNA extraction procedure. In certain embodiments of the invention the sample comprises a crude buffy coat fraction of whole blood. In certain other embodiments of the invention, the method further comprises the step of determining the ApoE genotype of the first subject and correlating said genotype with the risk or presence of disease. In some embodiments of the invention, the disease associated with altered mitochondrial function may be Alzheimer's Disease, Huntington's Disease, Parkinson's Disease, dystonia, schizophrenia, non-insulin dependent diabetes mellitus, mitochondrial encephalopathy, lactic acidosis, and stroke, myoclonic epilepsy ragged red fiber syndrome, and Leber's hereditary optic neuropathy.

Another aspect of the invention provides a method for quantifying extramitochondrial DNA, comprising: contacting a sample containing extramitochondrial DNA with an oligonucleotide primer having a nucleotide sequence complementary to at least a portion of the extramitochondrial DNA under conditions and for a time sufficient to allow hybridization of the primer to the extramitochondrial DNA; and detecting hybridization of the primer to the extramitochondrial DNA, and therefrom quantifying the extramitochondrial DNA.

It is another aspect of the invention to provide a method for quantifying extramitochondrial DNA, comprising: contacting a sample containing extramitochondrial DNA with an oligonucleotide primer having a nucleotide sequence complementary to at least a portion of the extramitochondrial DNA under conditions and for a time sufficient to allow hybridization of the primer to the extramitochondrial DNA; and detecting hybridization and extension of the primer to the extramitochondrial DNA to produce a product, and therefrom quantifying the extramitochondrial DNA.

Another aspect of the invention provides a method for quantifying extramitochondrial DNA, comprising: contacting a sample containing amplified extramitochondrial DNA with an oligonucleotide primer having a nucleotide sequence complementary to at least a portion of the extramitochondrial DNA under conditions and for a time sufficient to allow hybridization of the primer to the extramitochondrial DNA; and detecting hybridization of the primer to the extramitochondrial DNA, therefrom quantifying the extramitochondrial DNA.

In yet another aspect of the invention, a method is provided for quantifying extramitochondrial DNA by contacting a sample containing amplified extramitochondrial DNA with an oligonucleotide primer having a nucleotide sequence complementary to at least a portion of the extramitochondrial DNA under conditions and for a time sufficient to allow hybridization of the primer to the extramitochondrial DNA; and detecting hybridization and extension of the primer to the extramitochondrial DNA to produce a product, and therefrom quantifying the extramitochondrial DNA.

In one embodiment the extramitochondrial DNA is amplified by polymerase chain reaction, transcriptional amplification systems or self-sustained sequence replication. In certain embodiments of the various aspects of the invention, a single oligonucleotide primer is used. In certain embodiments of the invention a primer extension assay is used. In certain embodiments of the invention, the step of detecting may be by polymerase chain reaction, primer extension assay, ligase chain reaction or restriction fragment length polymorphism analysis.

In certain embodiments of the invention, the ratio r for a biological sample is calculated by determining the presence in the sample of a nucleotide sequence that may be SEQ ID NO:1, a portion of SEQ ID NO:1, SEQ ID NO:3 or a portion of SEQ ID NO:3. In one embodiment the portion of the nucleotide sequence of SEQ ID NO:1 corresponds to a portion of the nucleotide sequence of SEQ ID NO:2 encoding a mitochondrial cytochrome c oxidase. In another embodiment the portion of SEQ ID NO:1 corresponds to a portion of a mitochondrial cytochrome c oxidase encoding sequence that may be portion of a cytochrome c oxidase 1 (CO1) encoding sequence or a portion of a cytochrome c oxidase 2 (CO2) encoding sequence. In still other embodiments, the portion of the nucleotide sequence of SEQ ID NO:1 corresponds to a portion of the nucleotide sequence of SEQ ID NO:2 encoding a mitochondrial ATP synthetase subunit. In other embodiments, the portion of SEQ ID NO:1 corresponds to a portion of a mitochondrial ATP synthetase subunit encoding sequence that may be a portion of a sequence encoding ATP synthetase subunit 6 or a portion of a sequence encoding ATP synthetase subunit 8.

In some embodiments the nucleotide sequence of SEQ ID NO:1 corresponds to a portion of SEQ ID NO:2 that may be a portion of a sequence encoding ND1, a sequence encoding a portion of ND2 or a sequence encoding a portion of CO3. In other embodiments, the portion of the nucleotide sequence of SEQ ID NO:3 corresponds to a portion of the nucleotide sequence of SEQ ID NO:2 encoding a mitochondrial ATP synthetase subunit, which in some embodiments may further be a portion of a sequence encoding ATP synthetase subunit 6 or a portion of a sequence encoding ATP synthetase subunit 8. In still other embodiments, the nucleotide sequence of SEQ ID NO:1 corresponds to a portion of the nucleotide sequence of SEQ ID NO:2 encoding a mitochondrial tRNA, while in yet other embodiments the portion of the nucleotide sequence of SEQ ID NO:3 corresponds to a portion of the nucleotide sequence of SEQ ID NO:2 encoding a mitochondrial tRNA.

In another aspect the invention provides an isolated nucleic acid comprising all or a portion of the nucleotide sequence of SEQ ID NO:1 or a complementary sequence thereto. In another aspect the invention provides an isolated nucleic acid comprising all or a portion of a nucleotide sequence of SEQ ID NO:1 or a complementary sequence thereto, wherein the sequence of the isolated nucleic acid differs by at least one nucleotide from the corresponding sequence of a nucleic acid comprising the nucleotide sequence of SEQ ID NO:2 or a complementary sequence thereto. In another aspect the invention provides an isolated nucleic acid comprising all or a portion of the nucleotide sequence of SEQ ID NO:3 or a complementary sequence thereto. In another aspect the invention provides an isolated nucleic acid comprising all or a portion of a nucleotide sequence of SEQ ID NO:3 or a complementary sequence thereto, wherein the sequence of the isolated nucleic acid differs by at least one nucleotide from the corresponding sequence of a nucleic acid comprising the nucleotide sequence of SEQ ID NO:2 or a complementary sequence thereto.

In another aspect the invention provides a method for determining the risk or presence of a disease associated with altered mitochondrial function in a subject suspected of having or being at risk for having such a disease, by quantifying the amount of extramitochondrial DNA and the amount of mitochondrial DNA in a biological sample from the subject, and therefrom determining the risk or presence of the disease. It is another aspect of the invention to provide a method for determining the risk or presence of a disease associated with altered mitochondrial function in a first subject suspected of having or being at risk for having such a disease, by comparing the amount of extramitochondrial DNA and the amount of mitochondrial DNA in a biological sample from the first subject to the amount of extramitochondrial DNA and the amount of mitochondrial DNA in a biological sample from a second subject, and therefrom determining the risk or presence of the disease. In another aspect the invention provides a method for determining the risk or presence of a disease associated with altered mitochondrial function in a first subject suspected of having or being at risk for having such a disease, by quantifying the amount of extramitochondrial DNA and the amount of mitochondrial DNA in a biological sample from the subject and comparing the amount of extramitochondrial DNA and the amount of mitochondrial DNA to the amount of extramitochondrial DNA and the amount of mitochondrial DNA in a biological sample from a second subject known to be free of a risk or presence of a disease associated with altered mitochondrial function, and therefrom determining the risk or presence of the disease.

Another aspect of the invention provides a method of regulating a telomere by administering to a subject a nucleic acid molecule comprising all or a portion of SEQ ID NO:1 or a complementary portion thereto. In one embodiment, the administered nucleic acid molecule comprises an exmtDNA sequence. In another aspect, the invention provides a method of regulating a telomere by administering to a subject a nucleic acid molecule comprising all or a portion of SEQ ID NO:3 or a complementary portion thereto.

These and other aspects of the present invention will become evident upon reference to the following detailed description and attached drawings. In addition, various references are set forth herein which describe in more detail certain aspects of this invention, and are therefore incorporated by reference in their entirety.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the nucleotide sequence of SEQ ID NO:1.

FIG. 2 depicts the nucleotide sequence of SEQ ID NO:2 and amino acid sequences of SEQ ID NOs: 13-25.

FIG. 3 depicts the nucleotide sequence of SEQ ID NO:3 and human mtDNA sequence of SEQ ID NO:26.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed generally to compositions and methods for diagnosing the risk or presence of a disease associated with altered mitochondrial function in a subject, and to compositions and methods for the identification of agents that may be suitable for treating a disease associated with altered mitochondrial function. The invention utilizes a ratio, r, that may be useful for pharmacogenomic purposes, for example to stratify patient populations according to the suitability of particular therapeutic agents for use in such populations. The ratio r is the ratio of the amount of exmtDNA in a biological sample relative to the sum of the amount of exmtDNA plus mtDNA in the sample. As expressed quantitatively, the ratio r may be calculated using the formula:

r=x/(x+y)

wherein

x is the amount of exmtDNA in a sample, and

y is the amount of mtDNA in the sample.

In various aspects of the invention, as elaborated more fully herein, quantification of x and y provide, through calculation of r, parameters useful in diagnosis of a disease associated with altered mitochondrial function and in screening assays for agents that may be suitable for the treatment of such a disease.

As discussed above, “altered mitochondrial function” may refer to any condition or state, including those that accompany a disease, where any structure or activity that is directly or indirectly related to a mitochondrial function has been changed. Altered mitochondrial function may have its origin in extramitochondrial structures or events as well as in mitochondrial structures or events, in direct interactions between mitochondrial and extramitochondrial genes and/or their gene products, or in structural or functional changes that occur as the result of interactions between intermediates that may be formed as the result of such interactions, including metabolites, catabolites, substrates, precursors, cofactors and the like.

Also as discussed above, altered mitochondrial function may include (but need not be limited to) altered respiratory or metabolic activity in some or all cells of a biological source. For example, markedly impaired ETC activity may be an example of altered mitochondrial function, as may be generation of increased ROS or defective oxidative phosphorylation. As further examples, altered mitochondrial membrane potential, induction of apoptotic pathways and formation of atypical chemical and biochemical crosslinked species within a cell, whether by enzymatic or non-enzymatic mechanisms, may all be regarded as indicative of altered mitochondrial function. Without wishing to be bound by theory, alterations in the ratio r are believed to reflect chemical changes within affected cells that quantitatively influence recoveries of exmtDNA and/or mtDNA. For example, biochemical crosslinking events may result in the formation of DNA aggregates, DNA adducts or other molecular species that affect DNA recoveries following extraction procedures.

According to the present invention, alterations in the ratio r as defined above provide a novel and useful parameter for diagnosing the risk or presence of a disease associated with altered mitochondrial function in a subject, and for identifying agents that may be suitable for treating a disease associated with altered mitochondrial function. As discussed above, a number of diseases, including several degenerative diseases, are associated with alterations in mitochondrial function. Further, detection of an appropriate parameter of altered mitochondrial function can provide preclinical evidence for a risk of or predisposition to a disease.

Determination of the ratio r involves quantification of exmtDNA (x) and mtDNA (y) that may be based on strong but not necessarily absolute nucleotide sequence conservation when corresponding portions of mtDNA and exmtDNA are compared, as discussed herein. In most preferred embodiments of the invention, determination of r is accomplished by detecting minor nucleotide sequence differences in highly conserved mtDNA and exmtDNA regions, as elaborated below. The invention provides compositions and methods that include the use of nucleic acid molecules, or portions thereof, having nucleotide sequences that are found in the human mtDNA sequence SEQ ID NO:2 (Anderson et al., Nature 290:457, 1981) and fragments of SEQ ID NO:2 that are suitable for use as oligonucleotide primers in nucleic acid primer extension or amplification techniques, as hybridization probes for the detection of complementary nucleotide sequences in a sample or for any number of additional uses that are well known to those familiar with the art. ExmtDNA may be nuclear DNA, including chromosomal and non-chromosomal DNA, or non-nuclear extramitochondrial DNA that may be from any subcellular compartment, provided it is not mtDNA.

Nucleic acid sequences within the scope of the invention include isolated DNA and RNA sequences that specifically hybridize under conditions of moderate or high stringency to exmtDNA nucleotide sequences, including exmtDNA sequences disclosed herein or fragments thereof, and their complements. As used herein, conditions of moderate stringency, as known to those having ordinary skill in the art, and as defined by Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed. Vol. 1, pp. 1.101-104, Cold Spring Harbor Laboratory Press (1989), include use of a prewashing solution for the nitrocellulose filters 5×SSC, 0.5% SDS, 1.0 mM EDTA (pH 8.0), hybridization conditions of 50% formamide, 6×SSC at 42° C. (or other similar hybridization solution), and washing conditions of about 50-60° C., 0.5×SSC, 0.1% SDS. Conditions of high stringency are defined as hybridization conditions as above, and with washing at 60-68° C., 0.2×SSC, 0.1% SDS. In other embodiments, hybridization to an exmtDNA nucleotide sequence may be at normal stringency, which is approximately 25-30° C. below Tm of the native duplex (e.g., 5×SSPE, 0.5% SDS, 5×Denhardt's solution, 50% formamide, at 42° C. or equivalent conditions), at low stringency hybridizations, which utilize conditions approximately 40° C. below Tm, or at high stringency hybridizations, which utilize conditions approximately 10° C. below Tm. The skilled artisan will recognize that the temperature, salt concentration, and chaotrope composition of hybridization and wash solutions may be adjusted as necessary according to factors such as the length and nucleotide base composition of the probe. (See also, e.g., Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing, 1987.)

An “isolated nucleic acid molecule” refers to a polynucleotide molecule in the form of a separate fragment or as a component of a larger nucleic acid construct, that has been separated from its source cell (including the chromosome it normally resides in) at least once, preferably in a substantially pure form. Isolated nucleic acids may be nucleic acids having particular disclosed nucleotide sequences or may be regions, portions or fragments thereof. Those having ordinary skill in the art are able to prepare isolated nucleic acids having the complete nucleotide sequence, or the sequence of any portion of a particular isolated nucleic acid molecule, when provided with the appropriate nucleic acid sequence information as disclosed herein. Nucleic acid molecules may be comprised of a wide variety of nucleotides, including DNA, RNA, nucleotide analogues such as phosphorothioates or peptide nucleic acids, or other analogues with which those skilled in the art will be familiar, or some combination of these.

The present invention, as described herein, provides exmtDNA sequences and isolated exmtDNA nucleic acid molecules. exmtDNA may be isolated from genomic DNA, typically by first generating an appropriate DNA library through techniques for constructing libraries that are known in the art (see Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, 1989) or purchased from commercial sources (e.g., Clontech, Palo Alto, Calif.). Briefly, genomic DNA libraries can be constructed in chromosomal vectors, such as YACs (yeast artificial chromosomes), bacteriophage vectors, such as pBeloBAC11, λEMBL3, λgt10, cosmids, or plasmids. Alternatively, isolated exmtDNA may be prepared by preferentially amplifying exmtDNA sequences present in biological samples using, for example, DNA amplification methodologies such as PCR or other amplification techniques that are well known in the art, with suitable oligonucleotide primers complementary to exmtDNA sequences as disclosed herein.

In one embodiment, known mtDNA sequences derived from SEQ ID NO:2 (Anderson et al., Nature 290:457, 1981) may be utilized to design oligonucleotide hybridization probes suitable for screening genomic libraries. Preferably, such oligonucleotide probes are 18-30 bases in length and have sequences that, under the hybridization conditions selected, hybridize to complementary exmtDNA sequences lacking nucleotide substitutions, insertions or deletions (“mutations”) relative to the corresponding region of the mtDNA sequence of SEQ ID NO:2.

Portions of an exmtDNA sequence and the mtDNA sequence of SEQ ID NO:2 are regarded as “corresponding” nucleic acid sequences, regions, fragments or the like, based on the convention for numbering mtDNA nucleic acid positions according to SEQ ID NO:2 (Anderson et al., Nature 290:457, 1981), wherein an exmtDNA sequence is aligned with the mtDNA sequence of SEQ ID NO:2 such that at least 70%, preferably at least 80% and more preferably at least 90% of the nucleotides in a given sequence of at least 20 consecutive nt's of a sequence are identical. In certain preferred embodiments, an exmtDNA sequence is greater than 95% identical to a corresponding mtDNA sequence. In certain particularly preferred embodiments, an exmtDNA sequence is identical to a corresponding mtDNA sequence. Those oligonucleotide probes having sequences that are identical in corresponding regions of mtDNA and exmtDNA may be identified and selected following hybridization target DNA sequence analysis, to verify the absence of mutations in the target exmtDNA sequence relative to the primer mtDNA-derived sequence.

To facilitate hybridization detection, the oligonucleotide may be conveniently labeled, generally at the 5′ end, with a reporter molecule, such as a radionuclide, e.g., ³²P, enzymatic label, protein label, fluorescent label, biotin or other suitable labeling moieties known in the art. Such libraries are then generally plated as phage or colonies, depending upon the vector used. Subsequently, a plate replica to which the colonies or phage have been transferred, such as a nitrocellulose or nylon membrane or the like, is probed to identify candidate clones that contain the exmtDNA sequence. Such candidates may be verified as containing exmtDNA by any of various means including, for example, DNA sequence analysis or hybridization with a second, non-overlapping probe selected as described above to hybridize with target exmtDNA sequences lacking nucleotide substitutions, deletions or insertions relative to the corresponding portion of the mtDNA sequence of SEQ ID NO:2.

Once a library is identified as containing exmtDNA, the exmtDNA can be isolated by amplification. Briefly, when using genomic library DNA as a template, amplification primers are designed based upon known mtDNA sequences (SEQ ID NO:2) and primer “walking” is used to select primers that anneal to exmtDNA regions that are identical to mtDNA sequences. The primers preferably have a GC content of about 50% and contain restriction sites to facilitate cloning. Primers do not have self-complementary sequences, nor do they contain complementary sequences at their 3′ end (to prevent primer-dimer formation). The primers are annealed to genomic DNA and sufficient amplification cycles are performed to yield a product readily visualized by gel electrophoresis and staining. The amplified fragment is purified and inserted into a vector, such as λgt10 or pBS(M13+), and propagated. Confirmation of the nature of the fragment is obtained by DNA sequence analysis.

As an example of detection of mtDNA-like sequences in a DNA library, an oligonucleotide having a nucleotide sequence present in a portion of any human mtDNA gene, preferably one of the human mtDNA encoded genes NADH dehydrogenase subunit 1 (ND1), NADH dehydrogenase subunit 2 (ND2) or cytochrome c oxidase 3 (CO3) and more preferably one of the human mtDNA encoded genes CO, CO2, ATPase 8 or ATPase 6, may be labeled and used as a probe on a human genomic DNA library. An initial hybridization at normal stringency may yield candidate clones or fragments. If no hybridization is initially observed, varying degrees of stringency may be used. (See Sambrook et al., Ausubel et al., supra, and other well-known sources for stringency conditions.)

Where it is advantageous to use oligonucleotide primers according to the present invention, such primers may be 10-60 nucleotides in length, preferably 15-35 nucleotides and still more preferably 18-30 nucleotides in length. Primers as described above for use in isolating exmtDNA from genomic DNA may also be useful in the present invention for quantifying mtDNA and exmtDNA by any of a variety of techniques well known in the art for determining the amount of specific nucleic acid target sequences present in a sample based on specific hybridization of a primer to the target sequence. Optionally, in certain of these techniques, hybridization precedes nucleotide polymerase catalyzed extension of the primer using the strand containing the target sequence as a template, and/or ligation of oligonucleotides hybridized to adjacent target sequences, and embodiments of the invention using primer extension are particularly preferred. For examples of references on such quantitative detection techniques, including those that may be used to detect nucleotide insertions, substitutions or deletions in a portion of an exmtDNA sequence site near an oligonucleotide primer target hybridization site that corresponds to a portion of the mtDNA sequence of SEQ ID NO:2, and further including those that involve primer extension, see U.S. Pat. No. 5,760,205 and the references cited therein, all of which are hereby incorporated by reference, and see also, for example, Botstein et al. (Am. J. Hum. Gen. 32:314, 1980), Gibbs et al. (Nucl. Ac. Res. 17:2437, 1989), Newton et al. (Nucl. Ac. Res. 17:2503, 1989), Grossman et al. (Nucl. Ac. Res. 22:4527, 1994), and Saiki et al. (Proc. Nat. Acad. Sci. 86:6230, 1989), all of which are hereby incorporated by reference. A particularly useful method for this purpose is the primer extension assay disclosed by Fahy et al. (Nucl. Acids Res. 25:3102, 1997) and by Ghosh et al. (Am. J. Hum. Genet. 58:325, 1996), both of which references are hereby incorporated in their entireties, as is Krook et al. (Hum. Molec. Genet. 1:391, 1995) which teaches modification of primer extension reactions to detect multiple nucleotide substitutions, insertions, deletions or other mutations. Other examples of useful techniques for quantifying the presence of specific nucleic acid target sequences in a sample include but need not be limited to labeled probe hybridization to the target nucleic acid sequences with or without first partially separating target nucleic acids from other nucleic acids present in the sample.

Examples of other useful techniques for determining the amount of specific nucleic acid target sequences present in a sample based on specific hybridization of a primer to the target sequence include specific amplification of target nucleic acid sequences and quantification of amplification products, including but not limited to polymerase chain reaction (PCR, Gibbs et al., Nucl. Ac. Res. 17:2437, 1989), transcriptional amplification systems, strand displacement amplification and self-sustained sequence replication (3SR, Ghosh et al, in Molecular Methods for Virus Detection, 1995 Academic Press, NY, pp. 287-314), the cited references for which are hereby incorporated in their entireties. Examples of other useful techniques include ligase chain reaction, single stranded conformational polymorphism analysis, Q-beta replicase assay, restriction fragment length polymorphism (RFLP, Botstein et al., Am. J. Hum. Gen. 32:314, 1980) analysis and cycled probe technology, as well as other suitable methods that will be known to those familiar with the art.

In a particularly preferred embodiment of the invention, primer extension is used to quantify exmtDNA and mtDNA present in a biological sample. (Ghosh et al., Am. J. Hum. Genet. 58:325, 1996) This embodiment may offer certain advantages by permitting both exmtDNA and mtDNA to be simultaneously quantified using a single oligonucleotide primer capable of hybridizing to a complementary nucleic acid target sequence that is present in a defined region of mtDNA and in a corresponding region of a exmtDNA sequence. Without wishing to be bound by theory, the use of a single primer for quantification of exmtDNA and of mtDNA is believed to avoid uncertainties associated with potential disparities in the relative hybridization properties of multiple primers and may offer other advantages. Where such a target sequence is situated adjacent to an exmtDNA nucleotide sequence position that is a nucleotide substitution, insertion or deletion relative to the corresponding mtDNA sequence position, primer extension assays may be designed such that oligonucleotide extension products of primers hybridizing to mtDNA are of different lengths than oligonucleotide extension products of primers hybridizing to exmtDNA. Accordingly, the amount of exmtDNA in a sample and the amount of mtDNA in the sample may be determined by quantification of distinct extension products that are separable on the basis of sequence length or molecular mass, for purposes of calculating the ratio r as described above.

Sequence length or molecular mass of primer extension assay products may be determined using any known method for characterizing the size of nucleic acid sequences with which those skilled in the art are familiar. In a preferred embodiment, primer extension products are characterized by gel electrophoresis. In another preferred embodiment, primer extension products are characterized by mass spectrometry (MS), which may further include matrix assisted laser desorption ionization/time of flight (MALDI-TOF) analysis or other MS techniques known to those having skill in the art. See, for example, U.S. Pat. Nos. 5,622,824, 5,605,798 and 5,547,835, all of which are hereby incorporated by reference in their entireties. In another preferred embodiment, primer extension products are characterized by liquid or gas chromatography, which may further include high performance liquid chromatography (HPLC), gas chromatography-mass spectrometry (GC-MS) or other well known chromatographic methodologies.

Any exmtDNA sequence or portion of an exmtDNA sequence that corresponds to the human mtDNA sequence of SEQ ID NO:2 or a portion thereof or several portions thereof may be useful in this embodiment of the invention. Examples of human exmtDNA sequences that are useful in this and other embodiments of the invention are disclosed in SEQ ID NO:1 and in SEQ ID NO:3. Nucleotide positions at which exmtDNA and mtDNA differ are provided in Table 1 and Table 2, in which the identities of nucleotides at particular sequence positions in SEQ ID NO:2 and the corresponding positions in SEQ ID NO:1 and SEQ ID NO:3, respectively, are presented. Portions of SEQ ID NO:2 that include nucleic acid sequences encoding the mitochondrial ETC enzymes cytochrome c oxidase 1 (CO 1), cytochrome c oxidase 2 (CO 2), ATP synthetase subunit 8 (ATPase 8) and ATP synthetase subunit 6 (ATPase 6) may be particularly useful, and in preferred embodiments of the invention these sequences comprise isolated nucleic acid molecules that have nucleotide sequences identical or complementary to corresponding nucleic acid sequences present in exmtDNA of SEQ ID NO:1 and/or SEQ ID NO:3. Portions of SEQ ID NO:2 that include nucleid acid sequences encoding the mitochondrial tRNAs, including mitochondrial isoleucyl, glutaminyl, methionyl, aspartyl, cysteinyl, tyrosinyl and lysyl tRNAs may also be particularly useful, and in preferred embodiments of the invention these sequences comprise isolated nucleic acid molecules that have nucleotide sequences identical or complementary to corresponding nucleic acid sequences present in exmtDNA of SEQ ID NO:1 and/or SEQ ID NO:3.

TABLE 1 Human Human mt Gene Nucleotide mtDNA exmtDNA Region position SEQ ID NO:2 SEQ ID NO:1 NDI 4048 G A 4104 A G tRNA^(Ile) 4312 C T 4318 C T tRNA^(Gln) 4375 C G 4382 C A 4398 C T tRNA^(Met) 4456 C T ND2 4496 C T 4736 T C 4769 A G 4856 T C 4904 C T 4914 C T 4940 C T 4958 A G 4985 G A 4991 G A 5041 T C 5147 G A 5320 C T 5351 A G 5387 C T 5426 T C 5471 G A 5474 A G 5498 A G tRNA^(Cys) 5821 G A tRNA^(Tyr) 5840 C T CO1 6023 G A 6221 T C 6242 C T 6266 A C 6299 A G 6366 G A 6383 G A 6410 C T 6452 C T 6483 C T 6512 T C 6542 C T 6569 C A 6641 T C 6935 C T 6938 C T 7146 A G 7232 C T 7256 C T 7316 G A tRNA^(Asp) 7521 G A CO2 7650 C T 7705 T C 7810 C T 7868 C T 7891 C T 7912 G A 8021 A G 8065 G A 8140 C T 8152 G A 8167 T C 8196-8197 AC deletion 8203 C T ATPase8 8392 G A 8455 C T 8461 C T 8503 T C 8545 G A ATPase6 8545 G A 8655 C T 8677 A C 8701 A G 8718 A G 8860 A G 8943 C T 9060 C A 9075 C T 9103 C T 9168 C T 9175 C T CO3 9254 A G 9325 T C 9329 G C 9335 C T 9434 A G 9540 T C 9545 A G 9548 G A 9559 G C 9629 A G

TABLE 2 mt Gene Human mtDNA Human exmtDNA Region Nucleotide position SEQ ID NO:2 SEQ ID NO:3 tRNA^(Lys) 8310 T C 8311 T C 8336 T C 8345 C T 8348 A T 8349 C T 8351 C T ATPase8 8371 C A 8374 A G 8383 T C 8386 C T 8392 G A 8395 C T 8396 A G 8398 C T 8401 A C 8404 T C 8410 C A 8419 T C 8422 A G 8423 C T 8428 C T 8450 T C 8459 A C 8463 A G 8467 C T 8470 A G 8473 T C 8474 C A 8485 G A 8486 C T 8487 C T 8488 C T 8491 A T 8503 T C 8506 T C 8508 A G 8509 C T 8512 A G 8539 C T ATPase8/6 8541 G A 8557 G A 8562 C T 8566 A G 8568 C A ATPase6 8584 G A 8591 T C 8592 G A 8598 T C 8610 T C 8611 C T 8614 T C 8617 A G 8622 C A 8634 T C 8661 C T 8674 A C 8676 C T 8677 A C 8682 A C 8687 C T 8697 G A 8703 C G 8709 C T 8714 C T 8718 A G 8730 A G 8733 T C 8743 G A 8745 A T 8749 T C 8751 A G 8754 C T 8775 C T 8788 C T 8793 T C 8810 C T

In another particularly preferred embodiment of the invention, DNA in a biological sample containing exmtDNA and/or mtDNA is first amplified by methodologies well known in the art and described above, such that the amplification products may be used as templates in a method for quantifying the amount of exmtDNA and mtDNA present in the sample. Accordingly, it may be desirable to employ oligonucleotide primers that are complementary to target sequences that are identical in, and common to, mtDNA and exmtDNA, for example PCR amplification templates and primers prepared according to Fahy et al. (Nucl. Acids Res., 25:3102, 1997) and Davis et al. (Proc. Nat. Acad. Sci. USA 94:4526, 1997; see also Hirano et al., Proc. Nat. Acad. Sci. USA. 94:14894, 1997, and Wallace et al., Proc. Nat. Acad. Sci. USA 94:14900, 1997.)

Biological samples containing exmtDNA and mtDNA may comprise any tissue or cell preparation in which exmtDNA and mtDNA may be present. Biological samples may be provided by obtaining a blood sample, biopsy specimen, tissue explant, organ culture or any other tissue or cell preparation from a subject or a biological source. The subject or biological source may be a human or non-human animal, a primary cell culture or culture adapted cell line including but not limited to genetically engineered cell lines that may contain chromasomally integrated or episomal recombinant nucleic acid sequences, immortalized or immortalizable cell lines, somatic cell hybrid or cytoplasmic hybrid “cybrid” cell lines, differentiated or differentiatable cell lines, transformed cell lines and the like. In certain preferred embodiments of the invention, the subject or biological source may be suspected of having or being at risk for having a disease associated with altered mitochondrial function, and in certain preferred embodiments of the invention the subject or biological source may be known to be free of a risk or presence of such as disease.

In certain other preferred embodiments where it is desirable to determine whether or not a subject or biological source falls within clinical parameters indicative of Alzheimer's disease (AD), signs and symptoms of AD that are accepted by those skilled in the art may be used to so designate a subject or biological source, for example clinical signs referred to in McKhann et al. (Neurology 34:939, 1984, National Institute of Neurology, Communicative Disorders and Stroke and Alzheimer's Disease and Related Disorders Association Criteria of Probable AD, NINCDS-ADRDA) and references cited therein, or other means known in the art for diagnosing AD.

In certain aspects of the invention, biological samples containing mtDNA and exmtDNA may be obtained from the subject or biological source before and after contacting the subject or biological source with a candidate agent, for example to identify a candidate agent capable of effecting a change in the value of the ratio r, defined above, relative to the value of r before exposure of the subject or biological source to the agent.

In a most preferred embodiment of the invention, the biological sample containing mtDNA and exmtDNA may comprise a crude buffy coat fraction of whole blood, which is known in the art to comprise further a particulate fraction of whole blood enriched in white blood cells and platelets and substantially depleted of erythrocytes. Those familiar with the art will know how to prepare such a buffy coat fraction, which may be prepared by differential density sedimentation of blood components under defined conditions, including the use of density dependent separation media, or by other methods.

In another most preferred embodiment of the invention, the amount of exmtDNA and mtDNA in a biological sample may be quantified by first heating the sample in water to lyse cells contained therein, and then extracting cellular DNA from the lysed cells using an aqueous DNA extraction procedure. “Heating” may involve treating the cells for various times, typically 1-120 minutes, at a high temperature that is at least 80° C., preferably at least 90° C., more preferably at least 95° C. and most preferably in a boiling water bath. Based on the compositions and methods disclosed in the present application, the ordinarily skilled artisan will be able to readily determine optimal times and temperatures for heating samples to practice the invention without undue experimentation. As used herein, an “aqueous DNA extraction” method refers to preparation of DNA from such a boiled cell lysate without subjecting the lysate to sodium dodecylsulfate(SDS)/proteinase K treatments and/or without fractionating the lysate using a phenol-chloroform two-phase separation extraction step. Those skilled in the art will be familiar with various standard procedures for preparing and handling DNA without the use of SDS/ proteinase K and/or phenol-chloroform.

According to certain embodiments of the invention, the particular cell type or tissue type from which a biological sample is obtained may influence qualitative or quantitative aspects of the exmtDNA and/or mtDNA contained therein relative to exmtDNA and/or mtDNA obtained from distinct cell or tissue types of a common biological source. As described above, some diseases associated with altered mitochondrial function may manifest themselves in particular cell or tissue types. For example, AD is primarily a neurodegenerative disease that particularly effects changes in the central nervous system (CNS). It is therefore within the contemplation of the invention to quantify exmtDNA and mtDNA in biological samples from different cell or tissue types as may render the advantages of the invention most useful for a particular disease associated with altered mitochondrial function, and the relevant cell or tissue types will be known to those familiar with such diseases.

In order to determine whether a mitochondrial alteration may contribute to a particular disease state, it may be useful to construct a model system for diagnostic tests and for screening candidate therapeutic agents in which the nuclear genetic background may be held constant while the mitochondrial genome is modified. It is known in the art to deplete mitochondrial DNA from cultured cells to produce ρ⁰ cells, thereby preventing expression and replication of mitochondrial genes and inactivating mitochondrial function. See, for example, International Publication Number WO 95/26973, which is hereby incorporated by reference in its entirety, and references cited therein. It is further known in the art to repopulate such ρ⁰ cells with mitochondria derived from foreign cells in order to assess the contribution of the donor mitochondrial genotype to the respiratory phenotype of the recipient cells. Such cytoplasmic hybrid cells, containing genomic and mitochondrial DNAs of differing biological origins, are known as cybrids.

“ρ⁰ cells” are cells essentially completely depleted of mtDNA, and therefore have no functional mitochondrial respiration/electron transport activity. Such absence of mitochondrial respiration may be established by demonstrating a lack of oxygen consumption by intact cells in the absence of glucose, and/or by demonstrating a lack of catalytic activity of electron transport chain enzyme complexes having subunits encoded by mtDNA, using methods well known in the art. (See, e.g., Miller et al., J. Neurochem. 67:1897-1907, 1996.) That cells have become ρ⁰ cells may be further established by demonstrating that no mtDNA sequences are detectable within the cells. For example, using standard techniques well known to those familiar with the art, cellular mtDNA content may be measured using slot blot analysis of 1 μg total cellular DNA probed with a mtDNA-specific oligonucleotide probe radiolabeled with, e.g., ³²P to a specific activity ≧900 Ci/gm. Under these conditions ρ⁰ cells yield no detectable hybridizing probe signal. Alternatively, any other method known in the art for detecting the presence of mtDNA in a sample may be used that provides comparable sensitivity.

“Mitochondrial DNA depleted” cells (“mtDNA depleted cells”) are cells substantially but not completely depleted of functional mitochondria and/or mitochondrial DNA, by any method useful for this purpose. MtDNA depleted cells are preferably at least 80% depleted of mtDNA as measured using the slot blot assay described above for the determination of the presence of ρ⁰ cells, and more preferably at least 90% depleted of mtDNA. Most preferably, mtDNA depleted cells are depleted of >95% of their mtDNA.

Mitochondria to be transferred to construct model systems in accordance with the present invention may be isolated from virtually any tissue or cell source. Cell cultures of all types may potentially be used, as may cells from any tissue. However, fibroblasts, brain tissue, myoblasts and platelets are preferred sources of donor mitochondria. Platelets are the most preferred, in part because of their ready abundance, and their lack of nuclear DNA. This preference is not meant to constitute a limitation on the range of cell types that may be used as donor sources.

For example, platelets may be isolated by an adaptation of the method of Chomyn (Am. J. Hum. Genet. 54:966-974, 1994). However, it is not necessary that this particular method be used. Other methods are easily substituted. For instance, if nucleated cells are used, cell enucleation and isolation of mitochondria isolation can be performed as described by Chomyn et al., Mol. Cell. Biol. 11:2236-2244, 1991. Human tissue from a subject suspected of having or being at risk for having a disease associated with altered mitochondrial function, or from a subject known to be free of a risk or presence of such a disease, may be the source of donor mitochondrial DNA.

After preparation of mitochondria by isolation of platelets or enucleation of donor cells, the mitochondria may be transplanted into ρ⁰ cells or mtDNA depleted cells using any known technique for introducing an organelle into a recipient cell, including but not limited to polyethylene glycol (PEG) mediated cell membrane fusion, cell membrane permeabilization, cell-cytoplast fusion, virus mediated membrane fusion, liposome mediated fusion, particle mediated cellular uptake, microinjection or other methods known in the art. For example by way of illustration and not limitation, mitochondria donor cells (˜1×10⁷) are suspended in calcium-free Dulbecco's modified Eagle (DME) medium and mixed with ρ⁰ cells (˜0.5×10⁶) in a total volume of 2 ml for 5 minutes at room temperature. The cell mixture is pelleted by centrifugation and resuspended in 150 μl PEG (PEG 1000, J.T. Baker, Inc., 50% w/v in DME). After 1.5 minutes, the cell suspension is diluted with normal ρ⁰ cell medium containing pyruvate, uridine and glucose, and maintained in tissue culture plates. Medium is replenished daily, and after one week medium lacking pyruvate and uridine is used to inhibit growth of unfused ρ⁰ cells. These or other methods known in the art may be employed to produce cytoplasmic hybrid, or “cybrid”, cell lines.

As a non-limiting example, cybrid model systems may be useful for diagnosing a patient suspected of having or being at risk for a disease associated with altered mitochondrial function. According to this example, the patient's mitochondria are used to construct cybrid cells as described above. These cybrid cells may then be propagated in vitro and used to provide a biological sample for the determination of the ratio r, which can be compared to an r value calculated from samples of a control cybrid cell line constructed with mitochondria from a subject known to be free of disease. Where it may be desirable to compare the influence upon r of mitochondria from different sources, both cybrid cell lines may be constructed from the same ρ⁰ cell line to provide a constant background environment. These and similar uses of model systems according to the invention for determining the risk for or presence of a disease associated with altered mitochondrial function will be appreciated by those familiar with the art and are within the scope and spirit of the invention.

As another non-limiting example, cybrid model systems may be useful for identifying agents suitable for treating a disease associated with altered mitochondrial function. According to this example, a cybrid cell line may be a biological source in which the ratio r is calculated as described above, before and after cybrid cells are contacted with a candidate agent for treating disease. Such a cybrid cell line may be used to screen candidate agents by identifying those agents capable of effecting a change in the value of r relative to the value of r before exposure to the agent. The present invention thus provides model systems for selecting therapeutic agents that may be suitable for the treatment of diseases associated with altered mitochondrial function. These and similar uses of model systems according to the invention for the screening and identification of agents that influence the ratio r defined above, will be appreciated by those familiar with the art and are within the scope and spirit of the invention.

In addition, although the present invention is directed primarily towards model systems for diseases in which the mitochondria have metabolic alterations, it is not so limited. Conceivably there are disorders wherein mitochondria contain structural or morphological defects or anomalies, and the model systems of the present invention are of value, for example, to find drugs that can address that particular aspect of the disease. Also, there are certain individuals that have or are suspected of having extraordinarily effective or efficient mitochondrial function, and the model systems of the present invention may be of value in studying such mitochondria. Moreover, it may be desirable to put known normal mitochondria into cell lines having disease characteristics, in order to rule out the possibility that mitochondrial alterations contribute to pathogenesis. All of these and similar uses are within the scope of the present invention, and the use of the phrase “mitochondrial alteration” herein should not be construed to exclude such embodiments.

According to the present invention, a ratio r as defined herein is determined in a biological sample, for example by calculation following quantification of mtDNA and exmtDNA using a technique based on specific oligonucleotide hybridization to a target sequence. This hybridization may be optionally followed by target template directed extension, such as in primer extension assays described herein. For certain diseases associated with altered mitochondrial function, calculation of r may have diagnostic usefulness. For example, where other clinical indicators of a disease associated with altered mitochondrial function are known, values for r in subjects known to be free of a risk or presence of such disease based on the absence of these indicators may be determined to establish a control range for r. The ratio may also be calculated in biological samples obtained from subjects suspected of having or being at risk for having a disease associated with altered mitochondrial function, and compared to the control range of r values determined in disease free subjects. Those having familiarity with the art will appreciate that there may be any number of variations on the particular subjects, biological sources and bases for comparing r values that are useful beyond those that are expressly presented herein, and these additional uses are within the scope and spirit of the invention.

For instance, determination of r in may take the form of a diagnostic assay performed on whole blood collected from a subject by routine venous blood draw, on buffy coat cells prepared from blood or on biological samples that are other cells, organs or tissue from a subject. Alternatively, in certain situations it may be desirable to construct cybrid cell lines using mitochondria from either control subjects or subjects suspected of being at risk for a disease associated with altered mitochondrial function. Such cybrids may be used to determine r for diagnostic purposes, or as biological sources for screening assays to identify agents that may be suitable for treating disease based on their ability to change the r value obtained from treated cells. In one embodiment of this aspect of the invention, therapeutic agents or combinations of agents that are tailored to effectively treat an individual patient's particular disease may be identified by routine screening of candidate agents on cybrid cells constructed with the patient's mitochondria.

The present invention provides compositions and methods that are useful in pharmacogenomics, for the classification and/or stratification of a subject or a patient population, for instance correlation of one or more traits in a subject with indicators of the responsiveness to, or efficacy of, a particular therapeutic treatment. In one aspect of the invention, measurement of r in a biological sample from a subject is combined with identification of the subject's apolipoprotein E (APOE) genotype to determine the risk for, or presence of, Alzheimer's disease (AD) in the subject. The apolipoprotein E type 4 allele (APOE-ε4) allele is a genetic susceptibility factor for sporadic AD and confers a two fold risk for AD (Corder et al., Science 261:921, 1993; see also “National Institute on Aging/Alzheimer's Association Working Group Consensus Statement,” Lancet 347:1091, 1996 and references cited therein, all of which are hereby incorporated by reference in their entireties.). Accordingly, in a preferred embodiment of the invention, the method for determining the risk for or presence of AD in a subject by comparing r values will further comprise determining the APOE genotype of the subject suspected of being at risk for AD. By using the combination of the methods for determining r, as disclosed herein, and methods known in the art for determining APOE genotype, an enhanced ability to detect the relative risk for AD is provided by the instant invention along with other related advantages. Similarly, where APOE genotype and risk for AD are correlated, the present invention provides advantageous methods for identifying agents suitable for treating AD where such agents affect r in a biological source.

As described herein, determination of r may be used to stratify an AD patient population. Accordingly, in another preferred embodiment of the invention, determination of r in a biological sample from an AD subject may provide a useful correlative indicator for that subject. An AD subject so classified on the basis of an r value may then be monitored using AD clinical parameters referred to above, such that correlation between r value and any particular clinical score used to evaluate AD may be monitored. For example, stratification of an AD patient population according to r values may provide a useful marker with which to correlate the efficacy of any candidate therapeutic agent being used in AD subjects. In a further preferred embodiment of this aspect of the invention, determination of r in concert with determination of an AD subject's APOE genotype may also be useful. These and related advantages will be appreciated by those familiar with the art.

In another aspect, the invention provides exmtDNA sequences that may be useful in the detection or regulation of telomeric events that are related to diseases, including diseases associated with altered mitochondrial function, or in the identification of agents that are suitable for the treatment of such diseases. Dynamic processes in the telomeric regions of chromosomes that involve specific nucleic acid sequences, and in particular that may involve particular nucleotide polymerase and nuclease activities, have been implicated in chromosomal events that may be related to cellular and molecular mechanisms of disease. See, for example, Fossel, J. Amer. Med. Assoc. 279:1732 (1998); LaBranche et al., Nat. Genet. 19:199 (1998); Shay, Cancer J. Sci. Am. 4:526 (1998); Nowak et al., Cancer J. Sci. Am. 4:148 (1998); Iwama et al., Hum. Genet. 102:397 (1998), all of which are hereby incorporated by reference. In one embodiment of the invention, nucleic acid sequences are provided that may be used to monitor telomeric events, including but not limited to telomerase activity. As disclosed herein, nucleic acids having exmtDNA sequences may be used to increase or decrease telomeric processes, for instance by destabilizing or stabilizing telomers. Without wishing to be bound by theory, because telomeric structure is related to cellular growth potential and/or senescence, nucleic acid based intervention in regulation of telomeric structure may provide effective means for the detection or treatment of related disease processes. The present invention provides identification of human exmtDNA sequences in human chromosomal telomeric regions, and other related advantages.

The following examples are offered by way of illustration, and not by way of limitation.

EXAMPLES Example 1 Cloning of exmtDNA from Peripheral Blood Lymphocyte Genomic DNA Library

Peripheral blood lymphocytes were separated from the peripheral blood mononuclear cell fraction of freshly drawn venous blood from healthy human volunteers and the DNA extracted by standard techniques. Plasmid isolation, production of competent cells, transformation and manipulations using cloning vectors were performed essentially as described (Sambrook et al., Molecular Cloning, a Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989). The human lymphocyte DNA was partially digested with HindIII and inserted into the pBeloBAC11 vector (Genome Systems, Inc., St. Louis, Mo. with insert sizes ranging from 50 kb to over 240 kb to create a human genomic library. This library was screened by PCR using the following primers, which are complementary to mtDNA sequences in the CO1 encoding region of SEQ ID NO:2 but have single-base substitutions at the 3′ end. Details of PCR reaction conditions are provided in U.S. Pat. No. 5,565,323, which is hereby incorporated by reference, using 30 cycles instead of 25 cycles at 95° C. and with a single reaction being performed instead of five separate reactions.

5′-CCTTACACCTAGCAGGTA SEQ ID NO:4 5′-ACGCCGATGAATATGATAGC SEQ ID NO:5

A single positive clone was identified having a genomic DNA insert that included exmtDNA and was expanded, with the DNA then being purified using Magnum KB-100 columns (Genome Systems, St. Louis, Mo.). Portions of the insert were amplified with internal PCR primers complementary to human mtDNA sequences (Anderson et al., Nature 290:456, 1981) and sequenced using Prism DyeDeoxy terminator chemistry (Perkin-Elmer, Foster City, Calif.) according to the manufacturer's instructions. Sequence information at the junctions of exmtDNA and adjoining non-mtDNA in the positive pBeloBAC clone was obtained using the BigDye Terminator cycle sequencing kit (Perkin-Elmer) according to the supplier's recommendations. The products of sequencing reactions were purified by ethanol precipitation or by using CentriSep spin columns (Princeton Separations, Princeton, N.J.), then electrophoretically separated using an Applied Biosystems Model 373A DNA sequencing system (Applied Biosystems Division of Perkin-Elmer, Foster City, Calif.). Sequence Navigator software (Applied Biosystems) was used to analyze exmtDNA sequence data, and nucleotide insertions, deletions or substitutions were identified by comparing exmtDNA sequences to published human mtDNA sequence data. (SEQ ID NO:2, Anderson et al., Nature 290:456, 1981)

The insert contained the 5,844 base pair contiguous exmtDNA sequence of SEQ ID NO:1 (nucleotide positions 481-6337 in FIG. 1), which corresponded to and exhibited 98% sequence homology with nucleotide positions 3911-9755 of the human mtDNA sequence of SEQ ID NO:2. There were 89 nucleotide positions at which substitutions were detected, as indicated in Table 1. The complete ˜5.8 kb exmtDNA sequence is in a single reading frame relative to the corresponding region of the human mitochondrial genome (SEQ ID NO:2) with the exception of a two base pair deletion at nucleotide positions 8196-8197. Starting at the 5′ terminus (nt 481 in FIG. 1) and proceeding in the 3′ direction, the exmtDNA sequence includes DNA sequences corresponding, in order, to a truncated ND1 gene, complete ND2 and CO1 genes, a CO2 gene with the above noted two base pair deletion, complete ATP synthetase subunit 8 and ATP synthetase subunit 6 genes, and a truncated CO3 gene of the published human mtDNA sequence of SEQ ID NO:2 (Anderson et al., Nature 290:456, 1981). The non-mitochondrial DNA sequences on either side of the 5,844 base pair exmtDNA sequence (nucleotides 1-480 and 6338-6744) did not display homology to any nuclear DNA sequences listed in the GenBank database.

Example 2 Detection of exmtDNA in RHO-0 Cells

In order to verify that presumptive exmtDNA sequences originated from nuclear and not mitochondrial DNA present in the DNA preparation from which the human genomic library was constructed, two established cell lines were depleted of mtDNA using ethidium bromide to generate ρα cells (Miller et al., J. Neurochem. 67:1897, 1996) and assayed for the presence of exmtDNA sequences. Briefly, ρ0118/5 and 064/5 SH-SY5Y neuroblastoma cells and 0A431 epidermal carcinoma cells were produced and maintained as described (Miller et al., 1996). Cells were harvested and DNA was extracted with DNAzol (Molecular Research Center, Inc., Cincinnati, Ohio) according to the manufacturer's instructions. The recovered DNA was amplified by PCR and analyzed by primer extension assay using primers specific for a sequence region containing the nucleotide at position 7146 of the mtDNA sequence (SEQ ID NO:2), which corresponds to a nucleotide substitution in the exmtDNA sequence. (Table 1 and SEQ ID NO:1) PCR primers and reaction conditions and primer extension assays were as described in Fahy et al. (Nucl. Acids Res. 25:3102, 1997), which is hereby incorporated by reference in its entirety. Primer extension products corresponding to a region of the exmtDNA sequence of SEQ ID NO:1 and including a nucleotide corresponding to the guanosine residue at position 7146 or its complement were detected in p0 cells that contained no detectable mtDNA as described herein and in Miller et al. (J. Neurochem 67:1897, 1996).

Example 3 Absence of Detectable Transcripts of exmtDNA Sequences in RT-PCR Assay

The reverse transcription-polymerase chain reaction (RT-PCR; Rappolee et al., Science 241:708, 1991; Chelly et al., Nature 333:858, 1988; Brenner et al., BioTechniques 7:1096, 1989) technique was employed to determine whether the novel 5.8 kb exmtDNA sequence cloned from a human genomic DNA library is transcribed. The buffy coat fraction of freshly drawn human venous blood was prepared using Accuspin devices (Sigma, St. Louis, Mo.) according to the manufacturer's instructions and total RNA was extracted from isolated buffy coat cells with Trizol reagent (GibcoBRL, Bethesda, Md.) as recommended by the supplier. First strand cDNA was synthesized from poly-A+ mRNA using the SuperScript™ preamplification system (GibcoBRL) with oligo(dT) as primer according to the manufacturer's instructions. PCR was conducted using this cDNA as template and primers complementary to portions of the CO2 mtDNA sequence (SEQ ID NO:2) as described in Fahy et al. (Nucl. Ac. Res. 25:3102, 1997). The corresponding region of exmtDNA (SEQ ID NO:1) contains nucleotide substitutions at positions 7650 and 7868, relative to mtDNA (Table 1). Amplicons were purified and analyzed by the primer extension assay as described in Fahy et al. such that readily distinguishable products are predicted depending on whether or not the primer has hybridized to a target sequence adjacent to a sequence having the substitutions. Based on quantitative analysis of fluorescent band intensities of the primer extension products, mRNA encoding human CO2 gene products of mtDNA (SEQ ID NO:2) was detectable, but no MRNA encoding products from the corresponding exmtDNA region of SEQ ID NO: 1 was detected, indicating that the exmtDNA sequence is not expressed.

Example 4 Primer Extension Assay to Quantify exmtDNA and mtDNA

A competitive primer extension approach was used to simultaneously detect both mtDNA and exmtDNA sequences with a fluorophor-labeled primer and a selected mix of deoxynucleotides (dNTPs) and dideoxynucleotides (ddNTPs). The exmtDNA and mtDNA compete as templates in the primer extension reaction and are distinguished by differential extension of the primer. Thus, to determine the relative quantities of defmed portions of mtDNA (SEQ ID NO:2) and exmtDNA (SEQ ID NO:1) using the nucleotide substitution at position 7650 (Table 1) with the nucleotide combination of dATP, dTTP and ddGTP, the primer is extended by one base when the template is mtDNA. When exmtDNA is present as template, the primer is extended by three bases. The proportion of numtDNA in relation to mtDNA is estimated by comparing the ratio of fluorescence intensities of the gel-separated extension products with a standard curve generated from known mtDNA/numtDNA mixtures. (Fahy et al., Nucl. Ac. Res. 25:3102, 1997)

Thermo Sequenase™, dNTPs and ddNTPs were purchased from Amersham (Cleveland, Ohio). Calf intestine alkaline phosphatase and biochemical reagents were obtained from Boehringer Mannheim (Indianapolis, Ind.) and QIAquick PCR purification kits from Qiagen (Chatsworth, Calif.). Accuspin™ Tubes and HISTOPAQUE® 1077 were purchased from Sigma (St. Louis, Mo.) and EDTA vacutainers from Beckton Dickinson (San Jose, Calif.). UlTma™ DNA polymerase, AmpliTaq® DNA polymerase and reagents for DNA synthesis were purchased from Perkin Elmer (Foster City, Calif.). Oligonucleotides were synthesized on an Applied Biosystems 394 DNA/RNA synthesizer (Perkin Elmer) using standard phosphoramidite chemistry. 5′ Fluorescein-labeled oligonucleotide primers were obtained by using the 6-FAM Amidite reagent in the last step of the automated synthesis. Tritylated and fluorescein-labeled oligonucleotides were purified by reverse phase chromatography using an acetonitrile gradient in 0.1 M triethylammonium acetate, pH 6.8 running buffer. The oligonucleotides migrated as single bands on a 15% denaturing polyacrylamide gel. The homogeneity of the fluorescein-labeled oligonucleotides was independently assessed by electrophoresis on an Applied Biosystems Model 373 Sequencing System.

After IRB approval and informed consent, fresh venous blood samples were drawn from 837 patients with clinical diagnosis of probable Alzheimer's disease (AD mean age=74.7±1.1 years) and 191 controls (67±1 years; cognitively normal age-matched N=114; cortico-basal ganglionic degeneration N=2; Pick's disease N=1; Parkinson's disease N=24; non-insulin dependent diabetes mellitus N=29; insulin dependent diabetes mellitus N=6; Leigh's syndrome, N=2, Machado Joseph Disease N=2; idiopathic renal glycosuria N=1; progressive supranuclear palsy N=1; sporadic amyotropic lateral sclerosis N=6; familial sporadic amyotropic lateral sclerosis N=1; familial Alzheimer's disease N=2). AD patients met the National Institute of Neurological, Communicative Disorders and Stroke and Alzheimer's Diseases and Related Disorders Association (NINCDS-ADRDA) criteria of probable Alzheimer's disease (McKhann et al., Neurology 34:939, 1984).

Blood samples were collected in EDTA vacutainers and kept at 4° C. for no more than 24 h. The platelet/white blood cell fraction was isolated with Accuspin™ Tubes (Sigma Diagnostics, St. Louis, Mo.) using the following procedure. Three ml of HISTOPAQUE® 1077 (Sigma) were added to the upper chamber of each Accuspin™ Tube and the device was centrifuged at 1000 g for 30 s. Two to three ml of blood were then introduced into the upper chamber and separated by centrifugation at 1000 g for 10 min at room temperature. After centrifugation, the plasma and white blood cell layers were transferred to a new tube and the white blood cells were sedimented by centrifugation at 7,000 g for 10 min. The white cell pellet was resuspended in 0.4 ml of a solution containing 0.9% sodium chloride/1 mM EDTA and stored at −80° C. until use.

Frozen white blood cells (0.2 mL) were thawed and were sedimented by centrifugation at 12,000 g for 5 min. The white cell pellet was washed twice with 0.6 ml of Dulbecco's Phosphate Buffered Saline (PBS; GibcoBRL, Bethesda, Md.) and resuspended in 0.2 ml of water. The cells were lysed by incubation in a boiling water bath for 10 min. After cooling to room temperature, the cellular debris was sedimented by centrifugation at 14,000 g for 2 min. The supernatant was transferred to a new vial and the approximate concentration of the crude DNA preparation was estimated from its A₂₆₀ absorbance. The DNA sample was stored at −80° C.

Primer extension reaction templates were prepared by PCR amplification of cellular DNA. The reactions were carried out in a total volume of 50 μl using the primer pair sets described below. Following amplification, the PCR products were analyzed by electrophoresis on a 0.8% agarose gel. Reactions for analyzing exmtDNA to mtDNA ratios contained ˜1 μg of cellular DNA, 2.5 U of AmpliTaq® DNA polymerase, 20 pmol each of the light strand primer

5′-CATGCAGCGCAAGTAGGTCTACAAGAC-3′ (SEQ ID NO:6) and the heavy strand primer

5′-TGTTATGTAAAGGATGCGTAGGGATGG-3′ (SEQ ID NO:7)

and 10 nmol of each dNTP in PCR buffer (10 mM Tris. HCl, pH 8.3, 50 mM KCl, 2 mM MgCl₂). After an initial denaturation step at 95° C. for 10 s in a Gene Amp PCR System 9600 thermal cycler (Perkin Elmer), amplification was carried out for 30 cycles under the following conditions: 95° C. for 1 min, 60° C. for 1 min, 72° C. for 1 min. After the last cycle, reaction tubes were kept at 72° C. for 4 min to ensure extension of incomplete strands to the full length 255 base pair size product.

Residual nucleotides that persisted after the PCR reaction were dephosphorylated by adding 1 unit of calf intestine alkaline phosphatase (CAP) in 5 μL of 10×CAP buffer (100 mM Tris. HCl, pH 8.3, 10 mM MgCl₂, 10 mM ZnCl₂) to the PCR reaction mixture and incubating for 30 min at 37° C. in thermal cycler. Then 1.1 μL of 0.25 M EDTA, pH 8.0 was added and the alkaline phosphatase was denatured at 75° C. for 10 min.

Double-stranded PCR products were separated from primers, nucleosides and enzymes using QIAquick™ columns (Qiagen, Chatsworth, Calif.) and the buffers provided by the manufacturer. Thus, 250 μl of buffer PB were added to the PCR reaction mixture and mixed. A Qiaquick™ spin column (Qiagen) was placed in a 2 ml collection tube and the sample was loaded. The sample was centrifuged for 30-60 s at 14000 g and the flow through was discarded. The adsorbed PCR product was washed with 750 μl of buffer PE, and eluted with 50 μl of 10 mM Tris. HCl, pH 8.5. The purified product solution was dried in a Savant SpeedVac Concentrator and then reconstituted in 20 μl of water.

The fluorescein-labeled primer for analysis of the AD-associated mutation at nucleotide position 7650 was

TATGAGGGCGTGATCATGAAAG (SEQ ID NO:8)

using dATP and dTTP plus dideoxyGTP (ddGTP) to generate primer extension products of 23 and 25 nucleotides in length from mtDNA and exmtDNA templates, respectively.

Stock solutions of each dNTP and ddNTP were prepared by mixing equimolar amounts of the nucleotides with MgCl₂ and diluting the mixture to the desired concentration with 10 mM Tris, 1 mM EDTA, pH 8.0 (TE). The fluorescein-labeled primers were diluted in TE to provide final stock concentrations of 40 fmol/μl. One μl of the purified PCR-amplified DNA fragment was used as template for each assay.

Primer extension reactions were performed in a total volume of 8 μL. The thermostable enzyme, UlTma™ DNA polymerase (Perkin-Elmer, Foster City, Calif.) was used in primer extension assays for analyzing exmtDNA to mtDNA ratios. The reactions contained template, 20 fmol fluorescein-labeled primer, 400 μM ddNTPs/25 μM dNTPs of the appropriate nucleotide combination and 0.6 unit of enzyme in buffer containing 10 mM Tris-HCl, pH 8.8, 10 mM KCl, 0.002% Tween 20, 2 mM MgCl₂. Each set of primer extension assays included control reactions with mtDNA and exmtDNA templates.

The products of the primer extension reaction were analyzed on an ABI 373 Sequencer using a 12% denaturing polyacrylamide gel and Tris borate/EDTA as running buffer. Prior to electrophoresis, the samples in loading dye were denatured for 3 min at 85° C. Three μl aliquots of the standards (primer with no added template, reaction products from control DNA templates) and each unknown reaction mixture were then loaded and electrophoresed according the manufacturer's instructions. Fluorescent band intensities associated with the primer extension products were estimated by the GENESCAN™ 672 software program (Perkin Elmer, Applied Biosystems Division). Quantitative analysis was carried out by correlating the fluorescent band intensities of mtDNA and exmtDNA-derived extension products from unknown samples with those from mtDNA and exmtDNA control templates.

Example 5 Primer Extension Assay Using DNA Isolated from Affinity Isolated Mitochondria

Mitochondria were immunoaffinity isolated from cultured cells and then DNA was extracted from the isolated organelles, to determine whether detectable exmtDNA was present. Normal SH-SY5Y neuroblastoma cells, and normal and ρ⁰ A431 epidermal carcinoma cells were produced and maintained as described (Miller et al., J. Neurochem. 67:1897, 1996). Cells were harvested by scraping in MSB (0.21 M mannitol, 0.07 M sucrose, 0.05 M Tris-HCl, 0.01 M EDTA, pH 7.4; 10⁷ cells/ml) and lysed by three freeze-thaw cycles. Cellular debris was removed by centrifugation at 1000×g for 5 min. The mitochondria enriched supernatants were used for subsequent immunopurification steps. The buffy coat fraction of whole blood containing white blood cells and platelets as described above was also prepared and lysed by freeze-thaw as was done with the cultured cells, to produce a mitochondrial fraction for immunopurification.

Monoclonal anti-mitochondrial antibody MAB 1273 (Chemicon International, Temecula, Calif.) was added to the mitochondria enriched fractions for 2 hr. Antibody-mitochondria complexes were isolated using a secondary antibody bound to magnetic beads (Dynal Inc., Great Neck, N.Y.) according to the manufacturer's instructions. After extensive washing of the bead-bound antibody-mitochondria complexes with PBS/0.1% BSA, mtDNA was extracted from the complexes using DNAzol reagent (Molecular Research Center, Inc., Cincinnati, Ohio) according to the manufacturer's recommendations. Quantification of mtDNA and exmtDNA was performed using the competitive primer extension assay described above in Example 4. No DNA could be amplified from the DNA extracted from immunopurified mitochondria from the ρ⁰ A431 cell line, consistent with the depletion of mtDNA that is characteristic of the ρ⁰ state. Primer extension results indicated that no exmtDNA sequences were present in the DNA extracted from mitochondria of blood buffy coat cells, SH-SY5Y cells or A431 cells, confirming that exmtDNA sequences are of extramitochondrial origin.

Example 6 Determination of ApoE Genotype by Primer Extension Assay

Primer extension assay procedures, essentially as described above in Example 4 but with the modifications indicated here, were used to determine APOE genotype in a panel of 837 clinically diagnosed AD individuals and 191 control subjects (cognitively normal age matched individuals, patients with non-insulin dependent diabetes mellitus (NIDDM) and neurological controls). The APOE allele distribution in the AD population an controls in the study is shown in Table 3.

TABLE 3 APOE Genotype Distribution APOE Control AD allele (N = 191) (N = 837) 2/2 1 2/3 16 33 2/4 3 22 3/3 112 295 3/4 56 403 4/4 3 84

DNA isolation and preparation of primer extension reaction templates by PCR were essentially as described above in Example 4, except that for APOE analysis, a modification of the protocol of Livak and Hainer (1994) was used. Thus, for template preparation by PCR each reaction contained ˜1 μg of cellular DNA, 2.5 U of AmpliTaq® DNA polymerase, 20 pmol each of the forward primer

5′-GGCACGGCTGTCCAAGG-3′ (SEQ ID NO:9) and the reverse primer

5′-CCCGGCCTGGTACACTG-3′ (SEQ ID NO:10)

and 10 nmol of each dNTP in PCR buffer (10 mM Tris. HCI, pH 8.3, 50 mM KCl, 1.5 mM MgCl₂ supplemented with 5% DMSO). After an initial denaturation step at 95° C. for 10 s in a Gene Amp PCR System 9600 thermal cycler (Perkin Elmer), amplification was carried out for 25 cycles under the following conditions: 95° C. for 1 min, 55° C. for 1 min, 72° C. for 1 min. The PCR product obtained after amplification was 226 base pairs in length.

The fluorescein-labeled primers and nucleotide combinations for primer extension analysis of Apo-E alleles are shown in Table 4.

TABLE 4 Primer Extension Primer Product Gene Sequence (5′—>3′) size sizes dNTP ddNTP APOE GCGGACATGGAGGACGTG 18 19, 20 T G, C Codon (SEQ ID NO:11) 112 APOE CGATGCCGATGACCTGCAGAAG 22 23, 24 T G, C Codon (SEQ ID NO:12) 158

Thermo Sequenase™ (Amersham, Cleveland, Ohio) DNA polymerase catalyzed reactions for Apo-E allele analysis were performed with 20 fmol fluorescein-labeled primer, 25 μM each of the appropriate ddNTP/dNTP combination and 0.64 unit of enzyme in buffer containing 10 mM Tris-HCl, pH 9.5, 5 mM KCl, 0.002% Tween 20, 2 mM MgCl₂. Each set of primer extension assays included Apo-E allelic DNA controls. After an initial denaturation step at 95° C. for 2 min, the reaction conditions comprised 20 cycles of 95° C. for 20 s and 55° C. for 40 s. The samples were concentrated to ˜1 μl by heating open reaction tubes at 94° C. for 7 min. After the concentration step, 8 μl of loading dye (0.5% blue dextran in 83% formamide/8.3 mM EDTA, pH 8.0) was added.

The products of the primer extension reaction were analyzed as described above in Example 4. The Apo-E allele composition of unknown DNA samples was deduced by comparing the electrophoretic pattern of primer extension products with those from Apo-E allele DNA standards. APOE genotype distribution is shown in Table 3.

Example 7 Correlation of exmtDNA:mtDNA Ratio with Risk for AD

Primer extension assays as described in Example 4 were used to quantify the amounts of mtDNA and exmtDNA in blood samples from a panel of 837 clinically diagnosed AD individuals and 191 control subjects (cognitively normal age matched individuals, patients with non-insulin dependent diabetes mellitus (NIDDM) and neurological controls). For each subject, a ratio r was calculated using the formula:

r=x/(x+y)

wherein

x is the amount of exmtDNA in a sample, and

y is the amount of mtDNA in the sample.

The values for r were multiplied by 100 to give ranges of values according to which the patient population was stratified as shown in Table 5. Within each stratified range, the ratio of the number of subjects diagnosed with AD (McKhann et al., Neurology 34:939, 1984) to the number of subjects not having AD was determined, showing a correlation of increasing r value with increasing risk for AD (Table 5).

TABLE 5 Relative Risk for AD:by r Value r × 100 # of AD subjects:# of Non-AD subjects >15 1.42 >20 1.8 >25 2.2 >30 2.0

Example 8 Increased Predictive Value of exmtDNA:mtDNA Ratio in Subjects Having at Least One APoE4 Allele

Determination of the value r according to Example 7 was combined with determination of APOE genotype according to Example 6 using the patient populations characterized in Examples 6 and 7 to ascertain the relationship of APOE genotype to relative risk for AD, and to demonstrate the increased correlative value of APOE genotype combined with increasing r values with relative risk for AD. The relative risk for AD as a function of APOE genotype using the APOE alleles 2, 3 or 4 alone is shown in the bottom line of Table 6. The remainder of Table 6 shows the relative risk of AD as a function of APOE genotype and r value when subjects are stratified according to r as in Example 7. The relative risk for AD increases as a function of increasing r. In particular individuals with one APOE4 allele, and especially subjects who are homozygous for the APOE4 allele, exhibit increasing risk for AD as a function of increasing r, as shown in Table 6.

TABLE 6 Relative Risk for AD:by r value and ApoE Genotype (# AD:# Non-AD) ApoE Genotype r × 100 e2/3 e3/3 one e4 allele e3/4 e4/4 >15 1.5 1.5 2.2 2.1 7.1 >20 2.0 1.9 3.7 3.1 9.0 >25 1.7 1.8 5.3 3.0 >9.0 >30 2.5 2.0 4.6 3.0 >9.0 independent of r 0.5 0.6 1.9 1.7 6.3

Example 9 Identification of Agents that Alter VAlue of R

In this example, an agent suitable for treating AD is identified based on its ability to lower the value of r as defined above. A blood sample is obtained from a patient diagnosed with AD (McKhann et al., Neurology 34:939, 1984) and a ratio r is calculated using the formula:

r=x/(x+y)

wherein

x is the amount of exmtDNA in a sample, and

y is the amount of mtDNA in the sample, where x and y are determined

using the primer extension assay as described in Example 4. The candidate agent is then administered to the patient in a quantity and for a time sufficient to impart a therapeutically beneficial effect, and blood samples are periodically collected and processed using the primer extension assay as described in Example 4 to monitor alterations in the value r. Candidate agents are subjected to preliminary characterization for toxicity, bioavailability and modes of delivery prior to administration to a patient. An agent is selected that causes a reduction in the value for r, signifying an effect on the altered mitochondrial function in the patient that may underlie the differential extractability of exmtDNA and mtDNA that contributes to r values associated with increased risk for AD.

Example 10 Detection of exmtDNA Sequences in Telomeres

In this example, fluorescence in situ hybridization (FISH) is used to localize nucleic acid sequences that are present in SEQ ID NO:1 to the telomeric region of at least one human chromosome. The methods of Cannizzaro et al. (Methods Mol. Biol. 75:313, 1997) and references cited therein, all of which are incorporated hereby in their entireties, are used to conduct FISH. Human cell lines are grown to confluence on coverslips and then fixed and permeabilized for FISH analysis of metaphase chromosomes. The cloned pBeloBAC insert containing SEQ ID NO:1 as described above is excised from the plasmid vector and fluorescein labeled as described, and used to probe the fixed and permeabilized cells. Metaphase cells are evaluated by fluorescent laser scanning confocal microscopy and subchromosomal localization of the fluorescent probe to telomeric regions is observed. Cytological methods are used to prepare metaphase spreads of human peripheral blood leukocytes as described in the cited references, and the chromosomes are doubly labeled using suitable reporter moieties for discerning two signals, one being quinacrine to identify each chromosome by its characteristic banding pattern and the other being the labeled SEQ ID NO:1 insert, to correlate a particular chromosome with telomeric hybridization of the insert. The nucleic acid insert of SEQ ID NO:1 is next fragmented with restriction endonucleases, and the separated fragments are labeled to generate a panel of probes representing distinct sequence portions of SEQ ID NO:1, to determine whether the portion of the insert hybridizing to telomeres corresponds to an exmtDNA sequence or a non-exmtDNA sequence present in the insert as it was cloned from the human genomic library, as described above in Example 1.

From the foregoing, it will be appreciated that, although specific embodiments of the invention have been described herein for purposes of illustration, various modifications may be made without deviating from the spirit and scope of the invention. Accordingly, the invention is not limited except as by the appended claims.

26 6744 base pairs nucleic acid single linear 1 CCTACGGGCT ACTACAACCC TTCGCTGACG CCATAAAACT CTTCACCAAA GAGCCCCTAA 60 AACCCGCCAC ATCTACCATC ACCCTCTACA TCACCGCCCC GACCTTAGCT CTCACCATCG 120 CTCTTCTACT ATGAACCCCC CTCCCCATAC CCAACCCCCT GGTCAACCTC AACCTAGGCC 180 TCCTATTTAT TCTAGCCACC TCTAGCCTAG CCGTTTACTC AATCCTCTGA TCAGGGTGAG 240 CATCAAACTC AAACTACGCC CTGATCGGCG CACTGCGAGC AGTAGCCCAA ACAATCTCAT 300 ATGAAGTCAC CCTAGCCATC ATTCTACTAT CAACATTACT AATAAGTGGC TCCTTTAACC 360 TCTCCACCCT TATCACAACA CAAGAACACC TCTGATTACT CCTGCCATCA TGACCCTTGG 420 CCATAATATG ATTTATCTCC ACACTAGCAG AGACCAACCG AACCCCCTTC GACCTTGCCG 480 AAGGGGAGTC CGAACTAGTC TCAGGCTTCA ACATCGAATA CGCCGCAGGC CCCTTCGCCC 540 TATTCTTCAT AGCCGAATAC ACAAACATTA TTATAATAAA CACCCTCACC ACTACAATCT 600 TCCTAGGAAC AACATATRAC GCACTCTCCC CTGAACTCTA CACAACATAT TTTGTCACCA 660 AGACCCTACT TCTRACCTCC CTGTTCTTAT GAATTCGAAC AGCATACCCC CGATTCCGCT 720 ACGACCAACT CATACACCTC CTATGAAAAA ACTTCCTACC ACTCACCCTA GCATTACTTA 780 TATGATATGT CTCCATACCC ATTACAATCT CCAGCATTCC CCCTCAAACC TAAGAAATAT 840 GTCTGATAAA AGAGTTACTT TGATAGAGTA AATAATAGGA RYTTAAAYCC CCTTATTTCT 900 AGGACTATGA GAATCGAACC CATCCCTGAG AATCCAAAAT TCTCCGTGCC ACCTATCACA 960 CCCCATCCTA AAGTAAGGTC AGCTAAATAA GCTATCGGGC CCATACCCCG AAAATGTTGG 1020 TTATACCCTT CCCGTACTAA TTAATCCCCT GGCCCAACCC GTCATCTACT CTACCATCTT 1080 TGCAGGCACA CTCATCACAG CGCTAAGCTC GCACTGATTT TTTACCTGAG TAGGCCTAGA 1140 AATAAACATG CTAGCTTTTA TTCCAGTTCT AACCAAAAAA ATAAACCCTC GTTCCACAGA 1200 AGCTGCCATC AAGTATTTCC TCACGCAAGC AACCGCATCC ATAATCCTTC TAATAGCTAT 1260 CCTCTTCAAC AATATACTCT CCGGACAATG WRMCATWACC AATACYAYCA ATCAATACTC 1320 ATCATTAATA ATCATAATRG CTATAGCAAT AAAACTAGGA ATAGCCCCCT TTCACTTCTG 1380 AGTCCCAGAG GTTACCCAAG GGCACCCCTC TGACATCCGG CCTGCTYCTT CTCACATGAC 1440 AAAAACTAGC CCCCATCTCA ATCATATACC AAATYTCTCC CTCAYTAAAC GTAAGCCTTC 1500 TCCTCACTCT YTCAATCTTA TCCATCATRG CAGGCAGTTG AGGTGGATTA AACCARACCC 1560 ARCTACGCAA AATCTTAGCA TACTCCTCAA TTACCCACAT AGGATGAATA AYAGCAGTTC 1620 TACCGTACAA CCCTAACATA ACCATTCTTA ATTTAACTAT TTATATTATC CTAACTACTA 1680 CCGCATTCCT ACTACTCAAC TTAAACTCCA GCACCACAAC CCTACTACTA TCTCGCACCT 1740 GAAACAAGCT AACATGACTA ACACCCTTAA TTCCATCCAC CCTCCTCTCC CTAGGAGGCC 1800 TGCCCCCGCT AACCGGCTTT TTGCCCAAAT GGGCCATTAT CGAAGAATTC ACAAAAAACA 1860 ATAGCCTCAT CATCCCCACC ATCATAGCCA YCATCACCCT CCTTAACCTC TACTTCTACC 1920 TRCGCCTAAT CTACTCCACC TCAATCACAC TACTCCCYAT ATCTAACAAC GTAAAAATAA 1980 AATGACAGTT TGAACAYACA AAACCCACCC CATTCCTCCC CACACTCATC GCCCTTACCA 2040 CRCTRCTCCT ACCTATCTCC CCTTTTATRC TAATAATCTT ATAGAAATTT AGGTTAAATA 2100 CAGACCAAGA GCCTTCAAAG CCCTCAGTAA GTTGCAATAC TTAATTTCTG YAACAGCTAA 2160 GGACTGCAAA ACCCCACTCT GCATCAACTG NAACGCAAAT CAGCCACTTT AATTAAGCTT 2220 AAGCCCTTAC TAGACCAATG GGAACTTAAA CCCACAAACA CTTAGTTAAC AGCTAAGCAC 2280 CCTAATCAAC TGGCTTCAAT CTACTTCTCC CGCCGCCGGG AAAAAAGGCG GGAGAAGCCC 2340 CGGCAGGTTT GAAGCTGCTT CTTCGAATTT GCAATTCAAT ATGAAAATCA CCTCRGAGCT 2400 GGTAAAAAGA GGCYTAACCC CTGTCTTTAG ATTTACAGTC CAATGCTTCA CTCAGCCATT 2460 TTACCTCACC CCCACKGATG TTCGCCGACC GTTGACTATT CTCTACAAAC CACAAAGACA 2520 TTGGAACACT ATACCTATTA TTCGGCGCAT GAGCTGGAGT CCTAGGCACA GCTCTAAGCC 2580 TCCTTATTCG AGCCGARCTG GGCCAGCCAG GCAACCTTCT AGGTAACGAC CACATCTACA 2640 ACGTTATCGT CACAGCCCAT GCATTTGTAA TAATCTTCTT CATAGTAATA CCCATCATAA 2700 TCGGAGGCTT TGGCAACTGA CTAGTTCCCC TAATAATCGG TGCCCCCGAT ATGGCGTTTC 2760 CCCGCATAAA CAACATAAGC TTMTGACTCT TACCYCCCTC TCTCMTACTC CTGYTYGCAT 2820 CTGCTATAGT GGAGGCCGGM GCAGGAACAG GTTGAACAGT MTACCCTCCC TTRGCAGGGA 2880 ACTACTCCCA CCMTGGAGCC TCCGTAGACS TAACCATCTT STCCTTACAC YTAGCAGGTR 2940 TCTCCTTCTA TCTTAGGGGC CATCAATTTC ATCACAACAA TTATYAATAT AAAACCCCCT 3000 GCCATAACCC AATACCAAAC GCCCCTYTTC GTCTGATCCG TCCTAATCAC AGCAGTCYTA 3060 CTTCTCCTAT CTCTCCCAGT CCTAGCYGCT GGCATCACTA TACTACTAAC AGACCGYAMC 3120 YTCAACACCA CCTTYTTYGA CCCMGCCGGA GGAGGAGACC CCATTCTATA CCAACACCTA 3180 TTCTGATTTT TCGGTCACCC TGAAGTTTAT ATTCTYATCC TACCAGGCTT CGGAATAATC 3240 TCCCATATTG TAACTTACTA CTCCGGAAAA AAAGAACCAT TTGGATACAT AGGTATGGTC 3300 TGAGCTATGA TATCAATTGG CTTCCTAGGG TTTATCGTGT GAGCACACCA TATATTTACA 3360 GTAGGAATAG ACGTAGACAC ACGAGCATAT TTCACCTCCG CTACCATAAT CATCGCTATC 3420 CCCACCGGCG TCAAAGTATT TAGCTGACTC GCCACACTCC ACGGAAGCAA TATGAAATGA 3480 TCTGCTGCAG TGCTCTGAGC CCTAGGATTT ATTTTTCTTT TCACCGTAGG TGGCCTGACT 3540 GGCATTGTAT TAGCAAACTC ATCACTAGAC ATCGTACTAC ACGACACGTA CTACGTTGTA 3600 GCCCACTTCC ACTATGTCCT ATCAATAGGA GCTGTATTTG CCATCATAGG AGGCTTCATT 3660 CACTGATTTC CCCTATTCTC AGGGTACACC CTAGACCAAA CCTACGCCAA AATCCATTTC 3720 GCTATCATAT TCATCGGCGT AAATCTAACT TTCTTCCCAC AACACTTTCT CGGCCTATCC 3780 GGAATGCCCC GACGTTACTC GGACTAYCCC GATGCATACA CCACATGAAA YATCCTATCA 3840 TCTGTAGGCT CATTCATTTC TCTAACAGCA GTAATATTAA TAATTTTCAT AATTTGAGAA 3900 GCCTTCGCTT CGAAGCGAAA AGTCCTAATA GTAGAAGAAC CCTCCATAAA CCTGGAGTGA 3960 CTATATGGAT GCCCCCCACC CTACCACACA TTCGAAGAAC CCGTATACAT AAAATCTAGA 4020 CAAAAAAGGA AGGAATCGAA CCCCCCCAAA GCTGGTTTCA AGCCAACCCC ATGGCCTCCA 4080 TGACTTTTTC AAAAAGATAT TAGAAAAACC ATTTCATAAC TTTGTCAAAG TTAAATTATA 4140 GGCTAAATCC TATATATCTT AATGGCACAT GCAGCGCAAG TAGGTCTACA AGACGCTACT 4200 TCCCCTATCA TAGAAGAGCT TATCATCTTT CATGATCACG CCCTCATAAT CATTTTCCTT 4260 ATCTGCTTCC TAGTCCTGTA CGCCCTTTTC CTAACACTCA CAACAAAACT AACTAATACT 4320 AACATCTCAG ACGCTCAGGA AATAGAAACC GTCTGAACTA TCCTGCCCGC CATCATCCTA 4380 GTCCTYATCG CCCTCCCATY CCTACGCATC CTTTACATAA CAGACGAGGT CAACGATCCC 4440 TCCYTTACCA TCAAATCAAT TGGCCAYCAA TGGTACTGAA CCTACGARTA CACCGACTAC 4500 GGCGGACTAA TCTTCAACTC CTACATACTT CCCCCATTAT TCCTAGAACC AGGCGACCTG 4560 CGACTCCTTG ACGTTGACAA TCGAGTAGTA CTCCCGRTTG AAGCCCCCAT TCGTATAATA 4620 ATTACATCAC AAGACGTCTT ACACTCATGA GCTGTCCCCA CATTAGGCTT AAAAACAGAT 4680 GCAATTCCCG GACGTCTAAA CCAAACCACT TTCACTGCTA CACGACCAGG GGTATACTAC 4740 GGCCAATGCT CTGAAATCTG TGGAGCAAAC CAGTTTTATG CCCATCGTCC TAGAATTAAT 4800 TCCCCTAAAA ATCTTTGAAA TAGGGCCCGT ATTTACCCTA TAGCACCCCC TCTACCCCCT 4860 CTAGAGCCCA CTGTAAAGCT AACTTAGCAT TAACCTTTTA AGTTAAAGAT TAAGAGAACC 4920 AACACCTCTT TACAGTGAAA TGCCCCAACT AAATACTACC GTATGACCCA CCATAATTAC 4980 CCCCATACTC CTTACACTAT TCCTCATCAC CCAACTAAAA ATATTAAAYA CAAAYTACCA 5040 CCTACCTCCC TCACCAAAGC CCATAAAAAT AAAAAAYTAT AACAAACCCT GAGAACCAAA 5100 ATGAACGAAA ATCTGTTCRC TTCATTCATT GCCCCCACAA TCCTAGGCCT ACCCGCCGCA 5160 GTACTGATCA TTCTATTTCC CCCTCTATTG ATCCCCACCT CCAAATATCT CATCAACAAC 5220 CGACTAATTA CCACCCAACA ATGACTAATC MAACTAACCT CAAAACAAAT GATARCCATA 5280 CACAACACTA ARGGACGAAC CTGATCTCTT ATACTAGTAT CCTTAATCAT TTTTATTGCC 5340 ACAACTAACC TCCTCGGACT CCTGCCTCAC TCATTTACAC CAACCACCCA ACTATCTATA 5400 AACCTAGCCA TGGCCATCCC CTTATGAGCG GGCRCAGTGA TTATAGGCTT TCGCTCTAAG 5460 ATTAAAAATG CCCTAGCCCA CTTCTTACCA CAAGGCACAC CTACACCCCT TATCCCYATA 5520 CTAGTTATTA TCGAAACCAT CAGSCTAMTC ATTCAACCAA TAGCCCTGGC CGTAMGSCTA 5580 ACCGCTAACA TTACTGCAGG CCACCTAACT CATGMACCTA ATTGGAAGCG CCACMACTAG 5640 CAATATCAAS YATTAACCTT CCCTTCTACA CTTATCATYT TCACAATTCT AATTCTACTG 5700 ACTATCCTAG AAATCGCTGT CGCCTTAATC CAAGCCTACG TTTTYACACT TYTAGTAAGC 5760 CTCTACCTGC ACGACAACAC ATAATGACCC ACCAATCACA TGCCTATCAT ATAGTAAAAC 5820 CCAGCCCATG RCCCCTAACA GGGGCCCTCT CAGCCCTCCT AATGACCTCC GGCCTAGCCA 5880 TGTGATTTCA CTTCCACTCC AYAACSCTCC TYATACTAGG CCTACTAACC AACACACTAA 5940 CCATATACCA ATGATGGCGC GATGCTAACA CGAGTAAAGT CACATACCAA GGCCACCACA 6000 CACCACCTGT CCARAAAGGC CTTCGATACG GGATAATCCT ATTTATTACC TCAGAAGTTT 6060 TTTTCTTCGC AGGATTTTTC TGAGCCTTTT ACCACTCCAG CCTAGCTCCC TACCCCCCAA 6120 YTAGGRGGRC ACTGGCCCCS AACAGGCATC ACCCCGCTAA ATCCCCTAGA AGTCCCACTC 6180 CTAAACACAT CCGTATTACT CGCATCAGGR GTATCAATCA CCTGAGCTCA CCATAGTCTA 6240 ATAGAAAAAC AACCGAAACC AAATAATTCA AGCACTGCTT ATTACAATTT TACTGGGTCT 6300 CTATTTTACC CTCCTACAAG CCTCAGAGTA CTTCGAGGTT AAAATATTAG ATATTTCCCC 6360 TGATACAGGG CTCAATCTTT TTCTTTTTAA AGCAATATTT CTCAAAGTAC TTTTCACAGA 6420 ACTTAAGTTT CATTAAGCAC TTCACTAAAA GNAAAAGTCT GTGATCTAAT AAATTTGGAA 6480 AATATTGAGA ATTAGAGCCC CCTCTTAGAT ATGTACTGTA GCTACTCAGC TTGTTACAGA 6540 TGGAAGTAAA CATTGTAATA TTCACCCAGC TTTTGAGTGG ATGTCTATTA ACATCACCCA 6600 AATGAGTATT CCATGGAATG CACTTTGCAA AAACCTATTA TTCAAGAAAA ATTCTGGAGC 6660 ATGGAAAGCT ATTAATGGAT AAACCCATTC ACAAAATCAC ACCAAATATC TAAAATCATG 6720 TTTAAAATCT CCTAGAAATG GGTT 6744 16569 base pairs nucleic acid single linear 2 GATCACAGGT CTATCACCCT ATTAACCACT CACGGGAGCT CTCCATGCAT TTGGTATTTT 60 CGTCTGGGGG GTATGCACGC GATAGCATTG CGAGACGCTG GAGCCGGAGC ACCCTATGTC 120 GCAGTATCTG TCTTTGATTC CTGCCTCATC CTATTATTTA TCGCACCTAC GTTCAATATT 180 ACAGGCGAAC ATACTTACTA AAGTGTGTTA ATTAATTAAT GCTTGTAGGA CATAATAATA 240 ACAATTGAAT GTCTGCACAG CCACTTTCCA CACAGACATC ATAACAAAAA ATTTCCACCA 300 AACCCCCCCT CCCCCGCTTC TGGCCACAGC ACTTAAACAC ATCTCTGCCA AACCCCAAAA 360 ACAAAGAACC CTAACACCAG CCTAACCAGA TTTCAAATTT TATCTTTTGG CGGTATGCAC 420 TTTTAACAGT CACCCCCCAA CTAACACATT ATTTTCCCCT CCCACTCCCA TACTACTAAT 480 CTCATCAATA CAACCCCCGC CCATCCTACC CAGCACACAC ACACCGCTGC TAACCCCATA 540 CCCCGAACCA ACCAAACCCC AAAGACACCC CCCACAGTTT ATGTAGCTTA CCTCCTCAAA 600 GCAATACACT GAAAATGTTT AGACGGGCTC ACATCACCCC ATAAACAAAT AGGTTTGGTC 660 CTAGCCTTTC TATTAGCTCT TAGTAAGATT ACACATGCAA GCATCCCCGT TCCAGTGAGT 720 TCACCCTCTA AATCACCACG ATCAAAAGGG ACAAGCATCA AGCACGCAGC AATGCAGCTC 780 AAAACGCTTA GCCTAGCCAC ACCCCCACGG GAAACAGCAG TGATTAACCT TTAGCAATAA 840 ACGAAAGTTT AACTAAGCTA TACTAACCCC AGGGTTGGTC AATTTCGTGC CAGCCACCGC 900 GGTCACACGA TTAACCCAAG TCAATAGAAG CCGGCGTAAA GAGTGTTTTA GATCACCCCC 960 TCCCCAATAA AGCTAAAACT CACCTGAGTT GTAAAAAACT CCAGTTGACA CAAAATAGAC 1020 TACGAAAGTG GCTTTAACAT ATCTGAACAC ACAATAGCTA AGACCCAAAC TGGGATTAGA 1080 TACCCCACTA TGCTTAGCCC TAAACCTCAA CAGTTAAATC AACAAAACTG CTCGCCAGAA 1140 CACTACGAGC CACAGCTTAA AACTCAAAGG ACCTGGCGGT GCTTCATATC CCTCTAGAGG 1200 AGCCTGTTCT GTAATCGATA AACCCCGATC AACCTCACCA CCTCTTGCTC AGCCTATATA 1260 CCGCCATCTT CAGCAAACCC TGATGAAGGC TACAAAGTAA GCGCAAGTAC CCACGTAAAG 1320 ACGTTAGGTC AAGGTGTAGC CCATGAGGTG GCAAGAAATG GGCTACATTT TCTACCCCAG 1380 AAAACTACGA TAGCCCTTAT GAAACTTAAG GGTCGAAGGT GGATTTAGCA GTAAACTAAG 1440 AGTAGAGTGC TTAGTTGAAC AGGGCCCTGA AGCGCGTACA CACCGCCCGT CACCCTCCTC 1500 AAGTATACTT CAAAGGACAT TTAACTAAAA CCCCTACGCA TTTATATAGA GGAGACAAGT 1560 CGTAACATGG TAAGTGTACT GGAAAGTGCA CTTGGACGAA CCAGAGTGTA GCTTAACACA 1620 AAGCACCCAA CTTACACTTA GGAGATTTCA ACTTAACTTG ACCGCTCTGA GCTAAACCTA 1680 GCCCCAAACC CACTCCACCT TACTACCAGA CAACCTTAGC CAAACCATTT ACCCAAATAA 1740 AGTATAGGCG ATAGAAATTG AAACCTGGCG CAATAGATAT AGTACCGCAA GGGAAAGATG 1800 AAAAATTATA ACCAAGCATA ATATAGCAAG GACTAACCCC TATACCTTCT GCATAATGAA 1860 TTAACTAGAA ATAACTTTGC AAGGAGAGCC AAAGCTAAGA CCCCCGAAAC CAGACGAGCT 1920 ACCTAAGAAC AGCTAAAAGA GCACACCCGT CTATGTAGCA AAATAGTGGG AAGATTTATA 1980 GGTAGAGGCG ACAAACCTAC CGAGCCTGGT GATAGCTGGT TGTCCAAGAT AGAATCTTAG 2040 TTCAACTTTA AATTTGCCCA CAGAACCCTC TAAATCCCCT TGTAAATTTA ACTGTTAGTC 2100 CAAAGAGGAA CAGCTCTTTG GACACTAGGA AAAAACCTTG TAGAGAGAGT AAAAAATTTA 2160 ACACCCATAG TAGGCCTAAA AGCAGCCACC AATTAAGAAA GCGTTCAAGC TCAACACCCA 2220 CTACCTAAAA AATCCCAAAC ATATAACTGA ACTCCTCACA CCCAATTGGA CCAATCTATC 2280 ACCCTATAGA AGAACTAATG TTAGTATAAG TAACATGAAA ACATTCTCCT CCGCATAAGC 2340 CTGCGTCAGA TTAAAACACT GAACTGACAA TTAACAGCCC AATATCTACA ATCAACCAAC 2400 AAGTCATTAT TACCCTCACT GTCAACCCAA CACAGGCATG CTCATAAGGA AAGGTTAAAA 2460 AAAGTAAAAG GAACTCGGCA AATCTTACCC CGCCTGTTTA CCAAAAACAT CACCTCTAGC 2520 ATCACCAGTA TTAGAGGCAC CGCCTGCCCA GTGACACATG TTTAACGGCC GCGGTACCCT 2580 AACCGTGCAA AGGTAGCATA ATCACTTGTT CCTTAAATAG GGACCTGTAT GAATGGCTCC 2640 ACGAGGGTTC AGCTGTCTCT TACTTTTAAC CAGTGAAATT GACCTGCCCG TGAAGAGGCG 2700 GGCATAACAC AGCAAGACGA GAAGACCCTA TGGAGCTTTA ATTTATTAAT GCAAACAGTA 2760 CCTAACAAAC CCACAGGTCC TAAACTACCA AACCTGCATT AAAAATTTCG GTTGGGGCGA 2820 CCTCGGAGCA GAACCCAACC TCCGAGCAGT ACATGCTAAG ACTTCACCAG TCAAAGCGAA 2880 CTACTATACT CAATTGATCC AATAACTTGA CCAACGGAAC AAGTTACCCT AGGGATAACA 2940 GCGCAATCCT ATTCTAGAGT CCATATCAAC AATAGGGTTT ACGACCTCGA TGTTGGATCA 3000 GGACATCCCG ATGGTGCAGC CGCTATTAAA GGTTCGTTTG TTCAACGATT AAAGTCCTAC 3060 GTGATCTGAG TTCAGACCGG AGTAATCCAG GTCGGTTTCT ATCTACCTTC AAATTCCTCC 3120 CTGTACGAAA GGACAAGAGA AATAAGGCCT ACTTCACAAA GCGCCTTCCC CCGTAAATGA 3180 TATCATCTCA ACTTAGTATT ATACCCACAC CCACCCAAGA ACAGGGTTTG TTAAGATGGC 3240 AGAGCCCGGT AATCGCATAA AACTTAAAAC TTTACAGTCA GAGGTTCAAT TCCTCTTCTT 3300 AACAACATAC CCATGGCCAA CCTCCTACTC CTCATTGTAC CCATTCTAAT CGCAATGGCA 3360 TTCCTAATGC TTACCGAACG AAAAATTCTA GGCTATATAC AACTACGCAA AGGCCCCAAC 3420 GTTGTAGGCC CCTACGGGCT ACTACAACCC TTCGCTGACG CCATAAAACT CTTCACCAAA 3480 GAGCCCCTAA AACCCGCCAC ATCTACCATC ACCCTCTACA TCACCGCCCC GACCTTAGCT 3540 CTCACCATCG CTCTTCTACT ATGAACCCCC CTCCCCATAC CCAACCCCCT GGTCAACCTC 3600 AACCTAGGCC TCCTATTTAT TCTAGCCACC TCTAGCCTAG CCGTTTACTC AATCCTCTGA 3660 TCAGGGTGAG CATCAAACTC AAACTACGCC CTGATCGGCG CACTGCGAGC AGTAGCCCAA 3720 ACAATCTCAT ATGAAGTCAC CCTAGCCATC ATTCTACTAT CAACATTACT AATAAGTGGC 3780 TCCTTTAACC TCTCCACCCT TATCACAACA CAAGAACACC TCTGATTACT CCTGCCATCA 3840 TGACCCTTGG CCATAATATG ATTTATCTCC ACACTAGCAG AGACCAACCG AACCCCCTTC 3900 GACCTTGCCG AAGGGGAGTC CGAACTAGTC TCAGGCTTCA ACATCGAATA CGCCGCAGGC 3960 CCCTTCGCCC TATTCTTCAT AGCCGAATAC ACAAACATTA TTATAATAAA CACCCTCACC 4020 ACTACAATCT TCCTAGGAAC AACATATGAC GCACTCTCCC CTGAACTCTA CACAACATAT 4080 TTTGTCACCA AGACCCTACT TCTAACCTCC CTGTTCTTAT GAATTCGAAC AGCATACCCC 4140 CGATTCCGCT ACGACCAACT CATACACCTC CTATGAAAAA ACTTCCTACC ACTCACCCTA 4200 GCATTACTTA TATGATATGT CTCCATACCC ATTACAATCT CCAGCATTCC CCCTCAAACC 4260 TAAGAAATAT GTCTGATAAA AGAGTTACTT TGATAGAGTA AATAATAGGA GCTTAAACCC 4320 CCTTATTTCT AGGACTATGA GAATCGAACC CATCCCTGAG AATCCAAAAT TCTCCGTGCC 4380 ACCTATCACA CCCCATCCTA AAGTAAGGTC AGCTAAATAA GCTATCGGGC CCATACCCCG 4440 AAAATGTTGG TTATACCCTT CCCGTACTAA TTAATCCCCT GGCCCAACCC GTCATCTACT 4500 CTACCATCTT TGCAGGCACA CTCATCACAG CGCTAAGCTC GCACTGATTT TTTACCTGAG 4560 TAGGCCTAGA AATAAACATG CTAGCTTTTA TTCCAGTTCT AACCAAAAAA ATAAACCCTC 4620 GTTCCACAGA AGCTGCCATC AAGTATTTCC TCACGCAAGC AACCGCATCC ATAATCCTTC 4680 TAATAGCTAT CCTCTTCAAC AATATACTCT CCGGACAATG AACCATAACC AATACTACCA 4740 ATCAATACTC ATCATTAATA ATCATAATAG CTATAGCAAT AAAACTAGGA ATAGCCCCCT 4800 TTCACTTCTG AGTCCCAGAG GTTACCCAAG GCACCCCTCT GACATCCGGC CTGCTTCTTC 4860 TCACATGACA AAAACTAGCC CCCATCTCAA TCATATACCA AATCTCTCCC TCACTAAACG 4920 TAAGCCTTCT CCTCACTCTC TCAATCTTAT CCATCATAGC AGGCAGTTGA GGTGGATTAA 4980 ACCAGACCCA GCTACGCAAA ATCTTAGCAT ACTCCTCAAT TACCCACATA GGATGAATAA 5040 TAGCAGTTCT ACCGTACAAC CCTAACATAA CCATTCTTAA TTTAACTATT TATATTATCC 5100 TAACTACTAC CGCATTCCTA CTACTCAACT TAAACTCCAG CACCACGACC CTACTACTAT 5160 CTCGCACCTG AAACAAGCTA ACATGACTAA CACCCTTAAT TCCATCCACC CTCCTCTCCC 5220 TAGGAGGCCT GCCCCCGCTA ACCGGCTTTT TGCCCAAATG GGCCATTATC GAAGAATTCA 5280 CAAAAAACAA TAGCCTCATC ATCCCCACCA TCATAGCCAC CATCACCCTC CTTAACCTCT 5340 ACTTCTACCT ACGCCTAATC TACTCCACCT CAATCACACT ACTCCCCATA TCTAACAACG 5400 TAAAAATAAA ATGACAGTTT GAACATACAA AACCCACCCC ATTCCTCCCC ACACTCATCG 5460 CCCTTACCAC GCTACTCCTA CCTATCTCCC CTTTTATACT AATAATCTTA TAGAAATTTA 5520 GGTTAAATAC AGACCAAGAG CCTTCAAAGC CCTCAGTAAG TTGCAATACT TAATTTCTGT 5580 AACAGCTAAG GACTGCAAAA CCCCACTCTG CATCAACTGA ACGCAAATCA GCCACTTTAA 5640 TTAAGCTAAG CCCTTACTAG ACCAATGGGA CTTAAACCCA CAAACACTTA GTTAACAGCT 5700 AAGCACCCTA ATCAACTGGC TTCAATCTAC TTCTCCCGCC GCCGGGAAAA AAGGCGGGAG 5760 AAGCCCCGGC AGGTTTGAAG CTGCTTCTTC GAATTTGCAA TTCAATATGA AAATCACCTC 5820 GGAGCTGGTA AAAAGAGGCC TAACCCCTGT CTTTAGATTT ACAGTCCAAT GCTTCACTCA 5880 GCCATTTTAC CTCACCCCCA CTGATGTTCG CCGACCGTTG ACTATTCTCT ACAAACCACA 5940 AAGACATTGG AACACTATAC CTATTATTCG GCGCATGAGC TGGAGTCCTA GGCACAGCTC 6000 TAAGCCTCCT TATTCGAGCC GAGCTGGGCC AGCCAGGCAA CCTTCTAGGT AACGACCACA 6060 TCTACAACGT TATCGTCACA GCCCATGCAT TTGTAATAAT CTTCTTCATA GTAATACCCA 6120 TCATAATCGG AGGCTTTGGC AACTGACTAG TTCCCCTAAT AATCGGTGCC CCCGATATGG 6180 CGTTTCCCCG CATAAACAAC ATAAGCTTCT GACTCTTACC TCCCTCTCTC CTACTCCTGC 6240 TCGCATCTGC TATAGTGGAG GCCGGAGCAG GAACAGGTTG AACAGTCTAC CCTCCCTTAG 6300 CAGGGAACTA CTCCCACCCT GGAGCCTCCG TAGACCTAAC CATCTTCTCC TTACACCTAG 6360 CAGGTGTCTC CTCTATCTTA GGGGCCATCA ATTTCATCAC AACAATTATC AATATAAAAC 6420 CCCCTGCCAT AACCCAATAC CAAACGCCCC TCTTCGTCTG ATCCGTCCTA ATCACAGCAG 6480 TCCTACTTCT CCTATCTCTC CCAGTCCTAG CTGCTGGCAT CACTATACTA CTAACAGACC 6540 GCAACCTCAA CACCACCTTC TTCGACCCCG CCGGAGGAGG AGACCCCATT CTATACCAAC 6600 ACCTATTCTG ATTTTTCGGT CACCCTGAAG TTTATATTCT TATCCTACCA GGCTTCGGAA 6660 TAATCTCCCA TATTGTAACT TACTACTCCG GAAAAAAAGA ACCATTTGGA TACATAGGTA 6720 TGGTCTGAGC TATGATATCA ATTGGCTTCC TAGGGTTTAT CGTGTGAGCA CACCATATAT 6780 TTACAGTAGG AATAGACGTA GACACACGAG CATATTTCAC CTCCGCTACC ATAATCATCG 6840 CTATCCCCAC CGGCGTCAAA GTATTTAGCT GACTCGCCAC ACTCCACGGA AGCAATATGA 6900 AATGATCTGC TGCAGTGCTC TGAGCCCTAG GATTCATCTT TCTTTTCACC GTAGGTGGCC 6960 TGACTGGCAT TGTATTAGCA AACTCATCAC TAGACATCGT ACTACACGAC ACGTACTACG 7020 TTGTAGCCCA CTTCCACTAT GTCCTATCAA TAGGAGCTGT ATTTGCCATC ATAGGAGGCT 7080 TCATTCACTG ATTTCCCCTA TTCTCAGGCT ACACCCTAGA CCAAACCTAC GCCAAAATCC 7140 ATTTCACTAT CATATTCATC GGCGTAAATC TAACTTTCTT CCCACAACAC TTTCTCGGCC 7200 TATCCGGAAT GCCCCGACGT TACTCGGACT ACCCCGATGC ATACACCACA TGAAACATCC 7260 TATCATCTGT AGGCTCATTC ATTTCTCTAA CAGCAGTAAT ATTAATAATT TTCATGATTT 7320 GAGAAGCCTT CGCTTCGAAG CGAAAAGTCC TAATAGTAGA AGAACCCTCC ATAAACCTGG 7380 AGTGACTATA TGGATGCCCC CCACCCTACC ACACATTCGA AGAACCCGTA TACATAAAAT 7440 CTAGACAAAA AAGGAAGGAA TCGAACCCCC CAAAGCTGGT TTCAAGCCAA CCCCATGGCC 7500 TCCATGACTT TTTCAAAAAG GTATTAGAAA AACCATTTCA TAACTTTGTC AAAGTTAAAT 7560 TATAGGCTAA ATCCTATATA TCTTAATGGC ACATGCAGCG CAAGTAGGTC TACAAGACGC 7620 TACTTCCCCT ATCATAGAAG AGCTTATCAC CTTTCATGAT CACGCCCTCA TAATCATTTT 7680 CCTTATCTGC TTCCTAGTCC TGTATGCCCT TTTCCTAACA CTCACAACAA AACTAACTAA 7740 TACTAACATC TCAGACGCTC AGGAAATAGA AACCGTCTGA ACTATCCTGC CCGCCATCAT 7800 CCTAGTCCTC ATCGCCCTCC CATCCCTACG CATCCTTTAC ATAACAGACG AGGTCAACGA 7860 TCCCTCCCTT ACCATCAAAT CAATTGGCCA CCAATGGTAC TGAACCTACG AGTACACCGA 7920 CTACGGCGGA CTAATCTTCA ACTCCTACAT ACTTCCCCCA TTATTCCTAG AACCAGGCGA 7980 CCTGCGACTC CTTGACGTTG ACAATCGAGT AGTACTCCCG ATTGAAGCCC CCATTCGTAT 8040 AATAATTACA TCACAAGACG TCTTGCACTC ATGAGCTGTC CCCACATTAG GCTTAAAAAC 8100 AGATGCAATT CCCGGACGTC TAAACCAAAC CACTTTCACC GCTACACGAC CGGGGGTATA 8160 CTACGGTCAA TGCTCTGAAA TCTGTGGAGC AAACCACAGT TTCATGCCCA TCGTCCTAGA 8220 ATTAATTCCC CTAAAAATCT TTGAAATAGG GCCCGTATTT ACCCTATAGC ACCCCCTCTA 8280 CCCCCTCTAG AGCCCACTGT AAAGCTAACT TAGCATTAAC CTTTTAAGTT AAAGATTAAG 8340 AGAACCAACA CCTCTTTACA GTGAAATGCC CCAACTAAAT ACTACCGTAT GGCCCACCAT 8400 AATTACCCCC ATACTCCTTA CACTATTCCT CATCACCCAA CTAAAAATAT TAAACACAAA 8460 CTACCACCTA CCTCCCTCAC CAAAGCCCAT AAAAATAAAA AATTATAACA AACCCTGAGA 8520 ACCAAAATGA ACGAAAATCT GTTCGCTTCA TTCATTGCCC CCACAATCCT AGGCCTACCC 8580 GCCGCAGTAC TGATCATTCT ATTTCCCCCT CTATTGATCC CCACCTCCAA ATATCTCATC 8640 AACAACCGAC TAATCACCAC CCAACAATGA CTAATCAAAC TAACCTCAAA ACAAATGATA 8700 ACCATACACA ACACTAAAGG ACGAACCTGA TCTCTTATAC TAGTATCCTT AATCATTTTT 8760 ATTGCCACAA CTAACCTCCT CGGACTCCTG CCTCACTCAT TTACACCAAC CACCCAACTA 8820 TCTATAAACC TAGCCATGGC CATCCCCTTA TGAGCGGGCA CAGTGATTAT AGGCTTTCGC 8880 TCTAAGATTA AAAATGCCCT AGCCCACTTC TTACCACAAG GCACACCTAC ACCCCTTATC 8940 CCCATACTAG TTATTATCGA AACCATCAGC CTACTCATTC AACCAATAGC CCTGGCCGTA 9000 CGCCTAACCG CTAACATTAC TGCAGGCCAC CTACTCATGC ACCTAATTGG AAGCGCCACC 9060 CTAGCAATAT CAACCATTAA CCTTCCCTCT ACACTTATCA TCTTCACAAT TCTAATTCTA 9120 CTGACTATCC TAGAAATCGC TGTCGCCTTA ATCCAAGCCT ACGTTTTCAC ACTTCTAGTA 9180 AGCCTCTACC TGCACGACAA CACATAATGA CCCACCAATC ACATGCCTAT CATATAGTAA 9240 AACCCAGCCC ATGACCCCTA ACAGGGGCCC TCTCAGCCCT CCTAATGACC TCCGGCCTAG 9300 CCATGTGATT TCACTTCCAC TCCATAACGC TCCTCATACT AGGCCTACTA ACCAACACAC 9360 TAACCATATA CCAATGATGG CGCGATGTAA CACGAGAAAG CACATACCAA GGCCACCACA 9420 CACCACCTGT CCAAAAAGGC CTTCGATACG GGATAATCCT ATTTATTACC TCAGAAGTTT 9480 TTTTCTTCGC AGGATTTTTC TGAGCCTTTT ACCACTCCAG CCTAGCCCCT ACCCCCCAAT 9540 TAGGAGGGCA CTGGCCCCCA ACAGGCATCA CCCCGCTAAA TCCCCTAGAA GTCCCACTCC 9600 TAAACACATC CGTATTACTC GCATCAGGAG TATCAATCAC CTGAGCTCAC CATAGTCTAA 9660 TAGAAAACAA CCGAAACCAA ATAATTCAAG CACTGCTTAT TACAATTTTA CTGGGTCTCT 9720 ATTTTACCCT CCTACAAGCC TCAGAGTACT TCGAGTCTCC CTTCACCATT TCCGACGGCA 9780 TCTACGGCTC AACATTTTTT GTAGCCACAG GCTTCCACGG ACTTCACGTC ATTATTGGCT 9840 CAACTTTCCT CACTATCTGC TTCATCCGCC AACTAATATT TCACTTTACA TCCAAACATC 9900 ACTTTGGCTT CGAAGCCGCC GCCTGATACT GGCATTTTGT AGATGTGGTT TGACTATTTC 9960 TGTATGTCTC CATCTATTGA TGAGGGTCTT ACTCTTTTAG TATAAATAGT ACCGTTAACT 10020 TCCAATTAAC TAGTTTTGAC AACATTCAAA AAAGAGTAAT AAACTTCGCC TTAATTTTAA 10080 TAATCAACAC CCTCCTAGCC TTACTACTAA TAATTATTAC ATTTTGACTA CCACAACTCA 10140 ACGGCTACAT AGAAAAATCC ACCCCTTACG AGTGCGGCTT CGACCCTATA TCCCCCGCCC 10200 GCGTCCCTTT CTCCATAAAA TTCTTCTTAG TAGCTATTAC CTTCTTATTA TTTGATCTAG 10260 AAATTGCCCT CCTTTTACCC CTACCATGAG CCCTACAAAC AACTAACCTG CCACTAATAG 10320 TTATGTCATC CCTCTTATTA ATCATCATCC TAGCCCTAAG TCTGGCCTAT GAGTGACTAC 10380 AAAAAGGATT AGACTGAACC GAATTGGTAT ATAGTTTAAA CAAAACGAAT GATTTCGACT 10440 CATTAAATTA TGATAATCAT ATTTACCAAA TGCCCCTCAT TTACATAAAT ATTATACTAG 10500 CATTTACCAT CTCACTTCTA GGAATACTAG TATATCGCTC ACACCTCATA TCCTCCCTAC 10560 TATGCCTAGA AGGAATAATA CTATCGCTGT TCATTATAGC TACTCTCATA ACCCTCAACA 10620 CCCACTCCCT CTTAGCCAAT ATTGTGCCTA TTGCCATACT AGTCTTTGCC GCCTGCGAAG 10680 CAGCGGTGGG CCTAGCCCTA CTAGTCTCAA TCTCCAACAC ATATGGCCTA GACTACGTAC 10740 ATAACCTAAA CCTACTCCAA TGCTAAAACT AATCGTCCCA ACAATTATAT TACTACCACT 10800 GACATGACTT TCCAAAAAAC ACATAATTTG AATCAACACA ACCACCCACA GCCTAATTAT 10860 TAGCATCATC CCTCTACTAT TTTTTAACCA AATCAACAAC AACCTATTTA GCTGTTCCCC 10920 AACCTTTTCC TCCGACCCCC TAACAACCCC CCTCCTAATA CTAACTACCT GACTCCTACC 10980 CCTCACAATC ATGGCAAGCC AACGCCACTT ATCCAGTGAA CCACTATCAC GAAAAAAACT 11040 CTACCTCTCT ATACTAATCT CCCTACAAAT CTCCTTAATT ATAACATTCA CAGCCACAGA 11100 ACTAATCATA TTTTATATCT TCTTCGAAAC CACACTTATC CCCACCTTGG CTATCATCAC 11160 CCGATGAGGC AACCAGCCAG AACGCCTGAA CGCAGGCACA TACTTCCTAT TCTACACCCT 11220 AGTAGGCTCC CTTCCCCTAC TCATCGCACT AATTTACACT CACAACACCC TAGGCTCACT 11280 AAACATTCTA CTACTCACTC TCACTGCCCA AGAACTATCA AACTCCTGAG CCAATAACTT 11340 AATATGACTA GCTTACACAA TAGCTTTTAT AGTAAAGATA CCTCTTTACG GACTCCACTT 11400 ATGACTCCCT AAAGCCCATG TCGAAGCCCC CATCGCTGGG TCAATAGTAC TTGCCGCAGT 11460 ACTCTTAAAA CTAGGCGGCT ATGGTATAAT ACGCCTCACA CTCATTCTCA ACCCCCTGAC 11520 AAAACACATA GCCTACCCCT TCCTTGTACT ATCCCTATGA GGCATAATTA TAACAAGCTC 11580 CATCTGCCTA CGACAAACAG ACCTAAAATC GCTCATTGCA TACTCTTCAA TCAGCCACAT 11640 AGCCCTCGTA GTAACAGCCA TTCTCATCCA AACCCCCTGA AGCTTCACCG GCGCAGTCAT 11700 TCTCATAATC GCCCACGGGC TTACATCCTC ATTACTATTC TGCCTAGCAA ACTCAAACTA 11760 CGAACGCACT CACAGTCGCA TCATAATCCT CTCTCAAGGA CTTCAAACTC TACTCCCACT 11820 AATAGCTTTT TGATGACTTC TAGCAAGCCT CGCTAACCTC GCCTTACCCC CCACTATTAA 11880 CCTACTGGGA GAACTCTCTG TGCTAGTAAC CACGTTCTCC TGATCAAATA TCACTCTCCT 11940 ACTTACAGGA CTCAACATAC TAGTCACAGC CCTATACTCC CTCTACATAT TTACCACAAC 12000 ACAATGGGGC TCACTCACCC ACCACATTAA CAACATAAAA CCCTCATTCA CACGAGAAAA 12060 CACCCTCATG TTCATACACC TATCCCCCAT TCTCCTCCTA TCCCTCAACC CCGACATCAT 12120 TACCGGGTTT TCCTCTTGTA AATATAGTTT AACCAAAACA TCAGATTGTG AATCTGACAA 12180 CAGAGGCTTA CGACCCCTTA TTTACCGAGA AAGCTCACAA GAACTGCTAA CTCATGCCCC 12240 CATGTCTAAC AACATGGCTT TCTCAACTTT TAAAGGATAA CAGCTATCCA TTGGTCTTAG 12300 GCCCCAAAAA TTTTGGTGCA ACTCCAAATA AAAGTAATAA CCATGCACAC TACTATAACC 12360 ACCCTAACCC TGACTTCCCT AATTCCCCCC ATCCTTACCA CCCTCGTTAA CCCTAACAAA 12420 AAAAACTCAT ACCCCCATTA TGTAAAATCC ATTGTCGCAT CCACCTTTAT TATCAGTCTC 12480 TTCCCCACAA CAATATTCAT GTGCCTAGAC CAAGAAGTTA TTATCTCGAA CTGACACTGA 12540 GCCACAACCC AAACAACCCA GCTCTCCCTA AGCTTCAAAC TAGACTACTT CTCCATAATA 12600 TTCATCCCTG TAGCATTGTT CGTTACATGG TCCATCATAG AATTCTCACT GTGATATATA 12660 AACTCAGACC CAAACATTAA TCAGTTCTTC AAATATCTAC TCATCTTCCT AATTACCATA 12720 CTAATCTTAG TTACCGCTAA CAACCTATTC CAACTGTTCA TCGGCTGAGA GGGCGTAGGA 12780 ATTATATCCT TCTTGCTCAT CAGTTGATGA TACGCCCGAG CAGATGCCAA CACAGCAGCC 12840 ATTCAAGCAA TCCTATACAA CCGTATCGGC GATATCGGTT TCATCCTCGC CTTAGCATGA 12900 TTTATCCTAC ACTCCAACTC ATGAGACCCA CAACAAATAG CCCTTCTAAA CGCTAATCCA 12960 AGCCTCACCC CACTACTAGG CCTCCTCCTA GCAGCAGCAG GCAAATCAGC CCAATTAGGT 13020 CTCCACCCCT GACTCCCCTC AGCCATAGAA GGCCCCACCC CAGTCTCAGC CCTACTCCAC 13080 TCAAGCACTA TAGTTGTAGC AGGAATCTTC TTACTCATCC GCTTCCACCC CCTAGCAGAA 13140 AATAGCCCAC TAATCCAAAC TCTAACACTA TGCTTAGGCG CTATCACCAC TCTGTTCGCA 13200 GCAGTCTGCG CCCTTACACA AAATGACATC AAAAAAATCG TAGCCTTCTC CACTTCAAGT 13260 CAACTAGGAC TCATAATAGT TACAATCGGC ATCAACCAAC CACACCTAGC ATTCCTGCAC 13320 ATCTGTACCC ACGCCTTCTT CAAAGCCATA CTATTTATGT GCTCCGGGTC CATCATCCAC 13380 AACCTTAACA ATGAACAAGA TATTCGAAAA ATAGGAGGAC TACTCAAAAC CATACCTCTC 13440 ACTTCAACCT CCCTCACCAT TGGCAGCCTA GCATTAGCAG GAATACCTTT CCTCACAGGT 13500 TTCTACTCCA AAGACCACAT CATCGAAACC GCAAACATAT CATACACAAA CGCCTGAGCC 13560 CTATCTATTA CTCTCATCGC TACCTCCCTG ACAAGCGCCT ATAGCACTCG AATAATTCTT 13620 CTCACCCTAA CAGGTCAACC TCGCTTCCCC ACCCTTACTA ACATTAACGA AAATAACCCC 13680 ACCCTACTAA ACCCCATTAA ACGCCTGGCA GCCGGAAGCC TATTCGCAGG ATTTCTCATT 13740 ACTAACAACA TTTCCCCCGC ATCCCCCTTC CAAACAACAA TCCCCCTCTA CCTAAAACTC 13800 ACAGCCCTCG CTGTCACTTT CCTAGGACTT CTAACAGCCC TAGACCTCAA CTACCTAACC 13860 AACAAACTTA AAATAAAATC CCCACTATGC ACATTTTATT TCTCCAACAT ACTCGGATTC 13920 TACCCTAGCA TCACACACCG CACAATCCCC TATCTAGGCC TTCTTACGAG CCAAAACCTG 13980 CCCCTACTCC TCCTAGACCT AACCTGACTA GAAAAGCTAT TACCTAAAAC AATTTCACAG 14040 CACCAAATCT CCACCTCCAT CATCACCTCA ACCCAAAAAG GCATAATTAA ACTTTACTTC 14100 CTCTCTTTCT TCTTCCCACT CATCCTAACC CTACTCCTAA TCACATAACC TATTCCCCCG 14160 AGCAATCTCA ATTACAATAT ATACACCAAC AAACAATGTT CAACCAGTAA CTACTACTAA 14220 TCAACGCCCA TAATCATACA AAGCCCCCGC ACCAATAGGA TCCTCCCGAA TCAACCCTGA 14280 CCCCTCTCCT TCATAAATTA TTCAGCTTCC TACACTATTA AAGTTTACCA CAACCACCAC 14340 CCCATCATAC TCTTTCACCC ACAGCACCAA TCCTACCTCC ATCGCTAACC CCACTAAAAC 14400 ACTCACCAAG ACCTCAACCC CTGACCCCCA TGCCTCAGGA TACTCCTCAA TAGCCATCGC 14460 TGTAGTATAT CCAAAGACAA CCATCATTCC CCCTAAATAA ATTAAAAAAA CTATTAAACC 14520 CATATAACCT CCCCCAAAAT TCAGAATAAT AACACACCCG ACCACACCGC TAACAATCAA 14580 TACTAAACCC CCATAAATAG GAGAAGGCTT AGAAGAAAAC CCCACAAACC CCATTACTAA 14640 ACCCACACTC AACAGAAACA AAGCATACAT CATTATTCTC GCACGGACTA CAACCACGAC 14700 CAATGATATG AAAAACCATC GTTGTATTTC AACTACAAGA ACACCAATGA CCCCAATACG 14760 CAAAATTAAC CCCCTAATAA AATTAATTAA CCACTCATTC ATCGACCTCC CCACCCCATC 14820 CAACATCTCC GCATGATGAA ACTTCGGCTC ACTCCTTGGC GCCTGCCTGA TCCTCCAAAT 14880 CACCACAGGA CTATTCCTAG CCATGCACTA CTCACCAGAC GCCTCAACCG CCTTTTCATC 14940 AATCGCCCAC ATCACTCGAG ACGTAAATTA TGGCTGAATC ATCCGCTACC TTCACGCCAA 15000 TGGCGCCTCA ATATTCTTTA TCTGCCTCTT CCTACACATC GGGCGAGGCC TATATTACGG 15060 ATCATTTCTC TACTCAGAAA CCTGAAACAT CGGCATTATC CTCCTGCTTG CAACTATAGC 15120 AACAGCCTTC ATAGGCTATG TCCTCCCGTG AGGCCAAATA TCATTCTGAG GGGCCACAGT 15180 AATTACAAAC TTACTATCCG CCATCCCATA CATTGGGACA GACCTAGTTC AATGAATCTG 15240 AGGAGGCTAC TCAGTAGACA GTCCCACCCT CACACGATTC TTTACCTTTC ACTTCATCTT 15300 GCCCTTCATT ATTGCAGCCC TAGCAACACT CCACCTCCTA TTCTTGCACG AAACGGGATC 15360 AAACAACCCC CTAGGAATCA CCTCCCATTC CGATAAAATC ACCTTCCACC CTTACTACAC 15420 AATCAAAGAC GCCCTCGGCT TACTTCTCTT CCTTCTCTCC TTAATGACAT TAACACTATT 15480 CTCACCAGAC CTCCTAGGCG ACCCAGACAA TTATACCCTA GCCAACCCCT TAAACACCCC 15540 TCCCCACATC AAGCCCGAAT GATATTTCCT ATTCGCCTAC ACAATTCTCC GATCCGTCCC 15600 TAACAAACTA GGAGGCGTCC TTGCCCTATT ACTATCCATC CTCATCCTAG CAATAATCCC 15660 CATCCTCCAT ATATCCAAAC AACAAAGCAT AATATTTCGC CCACTAAGCC AATCACTTTA 15720 TTGACTCCTA GCCGCAGACC TCCTCATTCT AACCTGAATC GGAGGACAAC CAGTAAGCTA 15780 CCCTTTTACC ATCATTGGAC AAGTAGCATC CGTACTATAC TTCACAACAA TCCTAATCCT 15840 AATACCAACT ATCTCCCTAA TTGAAAACAA AATACTCAAA TGGGCCTGTC CTTGTAGTAT 15900 AAACTAATAC ACCAGTCTTG TAAACCGGAG ATGAAAACCT TTTTCCAAGG ACAAATCAGA 15960 GAAAAAGTCT TTAACTCCAC CATTAGCACC CAAAGCTAAG ATTCTAATTT AAACTATTCT 16020 CTGTTCTTTC ATGGGGAAGC AGATTTGGGT ACCACCCAAG TATTGACTCA CCCATCAACA 16080 ACCGCTATGT ATTTCGTACA TTACTGCCAG CCACCATGAA TATTGTACGG TACCATAAAT 16140 ACTTGACCAC CTGTAGTACA TAAAAACCCA ATCCACATCA AAACCCCCTC CCCATGCTTA 16200 CAAGCAAGTA CAGCAATCAA CCCTCAACTA TCACACATCA ACTGCAACTC CAAAGCCACC 16260 CCTCACCCAC TAGGATACCA ACAAACCTAC CCACCCTTAA CAGTACATAG TACATAAAGC 16320 CATTTACCGT ACATAGCACA TTACAGTCAA ATCCCTTCTC GTCCCCATGG ATGACCCCCC 16380 TCAGATAGGG GTCCCTTGAC CACCATCCTC CGTGAAATCA ATATCCCGCA CAAGAGTGCT 16440 ACTCTCCTCG CTCCGGGCCC ATAACACTTG GGGGTAGCTA AAGTGAACTG TATCCGACAT 16500 CTGGTTCCTA CTTCAGGGTC ATAAAGCCTA AATAGCCCAC ACGTTCCCCT TAAATAAGAC 16560 ATCACGATG 16569 823 base pairs nucleic acid single linear 3 AGAGAGAGGC ATGTGAATTG GGAATTTGGG AAAAATTTTT TGGGGGGAAG GAAAGAAATA 60 GAGGTCAAGA GGTAGAATAG AAGTTGATGA AGAAAAGAAA AAAAGAAGGT AATGAAGGGG 120 GTGCTGGATG TTTCCAACAC AAAGAAATGA TAAATGTTTG GGAGGATGGA TATTCTAATT 180 AGCCTAATTA GCCTGATTAG CCCTCGCCAG AGTTCACTGT AAAGCTAACC CAGCATTAAC 240 CTTTTAAGTT AAAGACTAAG AGAATCATTA TCTCTTTACA GTGAAATGCC ACAGCTAAAT 300 ACCACTGTAT GACCTGCTAT CATCACCCCA ATACTCCTCA CGTTATTTCT CATCACCCAA 360 CTAAAAATAC TAAACACACA CTGCCATCTG CCCACCTCAC CAAAATTTAT TAAAATAAAA 420 AACTACAGTA AGCCCTGAGA ACCAAAATGA ACGAAAATTT ATTCGCTTCA TTCATTACCC 480 CTACAGTACT AGGCCTACCC GCCACAGTAC CAATCATCCT ATTTCCCCCC TTACTGGTCC 540 CAACCTCCAA ATACCTCATC AACAACCGAC TAATCACCAC TCAACAATGA CTACTTCAAC 600 TCACCTTAAA ACAAATAATA ACGATACATA ACATTAAGGG ACGAACCTGG TCCCTTATAC 660 TAATTTCCCT GATTATTTTT ATTGCCACAA CTAATCTCCT CGGACTCTTG CCCCACTCAT 720 TTACACCAAT CACTATACAT GTGTCTATTG AAACGTCACT ATGTGTGCCC CATGAATATG 780 TACATATTAT TATGTGATGT ACATGATTAT GTACACATTA TGT 823 18 base pairs nucleic acid single linear 4 CCTTACACCT AGCAGGTA 18 20 base pairs nucleic acid single linear 5 ACGCCGATGA ATATGATAGC 20 27 base pairs nucleic acid single linear 6 CATGCAGCGC AAGTAGGTCT ACAAGAC 27 27 base pairs nucleic acid single linear 7 TGTTATGTAA AGGATGCGTA GGGATGG 27 22 base pairs nucleic acid single linear 8 TATGAGGGCG TGATCATGAA AG 22 17 base pairs nucleic acid single linear 9 GGCACGGCTG TCCAAGG 17 17 base pairs nucleic acid single linear 10 CCCGGCCTGG TACACTG 17 18 base pairs nucleic acid single linear 11 GCGGACATGG AGGACGTG 18 22 base pairs nucleic acid single linear 12 CGATGCCGAT GACCTGCAGA AG 22 318 amino acids amino acid linear 13 Met Pro Met Ala Asn Leu Leu Leu Leu Ile Val Pro Ile Leu Ile Ala 1 5 10 15 Met Ala Phe Leu Met Leu Thr Glu Arg Lys Ile Leu Gly Tyr Met Gln 20 25 30 Leu Arg Lys Gly Pro Asn Val Val Gly Pro Tyr Gly Leu Leu Gln Pro 35 40 45 Phe Ala Asp Ala Met Lys Leu Phe Thr Lys Glu Pro Leu Lys Pro Ala 50 55 60 Thr Ser Thr Ile Thr Leu Tyr Ile Thr Ala Pro Thr Leu Ala Leu Thr 65 70 75 80 Ile Ala Leu Leu Leu Trp Thr Pro Leu Pro Met Pro Asn Pro Leu Val 85 90 95 Asn Leu Asn Leu Gly Leu Leu Phe Ile Leu Ala Thr Ser Ser Leu Ala 100 105 110 Val Tyr Ser Ile Leu Trp Ser Gly Trp Ala Ser Asn Ser Asn Tyr Ala 115 120 125 Leu Ile Gly Ala Leu Arg Ala Val Ala Gln Thr Ile Ser Tyr Glu Val 130 135 140 Thr Leu Ala Ile Ile Leu Leu Ser Thr Leu Leu Met Ser Gly Ser Phe 145 150 155 160 Asn Leu Ser Thr Leu Ile Thr Thr Gln Glu His Leu Trp Leu Leu Leu 165 170 175 Pro Ser Trp Pro Leu Ala Met Met Trp Phe Ile Ser Thr Leu Ala Glu 180 185 190 Thr Asn Arg Thr Pro Phe Asp Leu Ala Glu Gly Glu Ser Glu Leu Val 195 200 205 Ser Gly Phe Asn Ile Glu Tyr Ala Ala Gly Pro Phe Ala Leu Phe Phe 210 215 220 Met Ala Glu Tyr Thr Asn Ile Ile Met Met Asn Thr Leu Thr Thr Thr 225 230 235 240 Ile Phe Leu Gly Thr Thr Tyr Asp Ala Leu Ser Pro Glu Leu Tyr Thr 245 250 255 Thr Tyr Phe Val Thr Lys Thr Leu Leu Leu Thr Ser Leu Phe Leu Trp 260 265 270 Ile Arg Thr Ala Tyr Pro Arg Phe Arg Tyr Asp Gln Leu Met His Leu 275 280 285 Leu Trp Lys Asn Phe Leu Pro Leu Thr Leu Ala Leu Leu Met Trp Tyr 290 295 300 Val Ser Met Pro Ile Thr Ile Ser Ser Ile Pro Pro Gln Thr 305 310 315 347 amino acids amino acid linear 14 Ile Asn Pro Leu Ala Gln Pro Val Ile Tyr Ser Thr Ile Phe Ala Gly 1 5 10 15 Thr Leu Ile Thr Ala Leu Ser Ser His Trp Phe Phe Thr Trp Val Gly 20 25 30 Leu Glu Met Asn Met Leu Ala Phe Ile Pro Val Leu Thr Lys Lys Met 35 40 45 Asn Pro Arg Ser Thr Glu Ala Ala Ile Lys Tyr Phe Leu Thr Gln Ala 50 55 60 Thr Ala Ser Met Ile Leu Leu Met Ala Ile Leu Phe Asn Asn Met Leu 65 70 75 80 Ser Gly Gln Trp Thr Met Thr Asn Thr Thr Asn Gln Tyr Ser Ser Leu 85 90 95 Met Ile Met Met Ala Met Ala Met Lys Leu Gly Met Ala Pro Phe His 100 105 110 Phe Trp Val Pro Glu Val Thr Gln Gly Thr Pro Leu Thr Ser Gly Leu 115 120 125 Leu Leu Leu Thr Trp Gln Lys Leu Ala Pro Ile Ser Ile Met Tyr Gln 130 135 140 Ile Ser Pro Ser Leu Asn Val Ser Leu Leu Leu Thr Leu Ser Ile Leu 145 150 155 160 Ser Ile Met Ala Gly Ser Trp Gly Gly Leu Asn Gln Thr Gln Leu Arg 165 170 175 Lys Ile Leu Ala Tyr Ser Ser Ile Thr His Met Gly Trp Met Met Ala 180 185 190 Val Leu Pro Tyr Asn Pro Asn Met Thr Ile Leu Asn Leu Thr Ile Tyr 195 200 205 Ile Ile Leu Thr Thr Thr Ala Phe Leu Leu Leu Asn Leu Asn Ser Ser 210 215 220 Thr Thr Thr Leu Leu Leu Ser Arg Thr Trp Asn Lys Leu Thr Trp Leu 225 230 235 240 Thr Pro Leu Ile Pro Ser Thr Leu Leu Ser Leu Gly Gly Leu Pro Pro 245 250 255 Leu Thr Gly Phe Leu Pro Lys Trp Ala Ile Ile Glu Glu Phe Thr Lys 260 265 270 Asn Asn Ser Leu Ile Ile Pro Thr Ile Met Ala Thr Ile Thr Leu Leu 275 280 285 Asn Leu Tyr Phe Tyr Leu Arg Leu Ile Tyr Ser Thr Ser Ile Thr Leu 290 295 300 Leu Pro Met Ser Asn Asn Val Lys Met Lys Trp Gln Phe Glu His Thr 305 310 315 320 Lys Pro Thr Pro Phe Leu Pro Thr Leu Ile Ala Leu Thr Thr Leu Leu 325 330 335 Leu Pro Ile Ser Pro Phe Met Leu Met Ile Leu 340 345 513 amino acids amino acid linear 15 Met Phe Ala Asp Arg Trp Leu Phe Ser Thr Asn His Lys Asp Ile Gly 1 5 10 15 Thr Leu Tyr Leu Leu Phe Gly Ala Trp Ala Gly Val Leu Gly Thr Ala 20 25 30 Leu Ser Leu Leu Ile Arg Ala Glu Leu Gly Gln Pro Gly Asn Leu Leu 35 40 45 Gly Asn Asp His Ile Tyr Asn Val Ile Val Thr Ala His Ala Phe Val 50 55 60 Met Ile Phe Phe Met Val Met Pro Ile Met Ile Gly Gly Phe Gly Asn 65 70 75 80 Trp Leu Val Pro Leu Met Ile Gly Ala Pro Asp Met Ala Phe Pro Arg 85 90 95 Met Asn Asn Met Ser Phe Trp Leu Leu Pro Pro Ser Leu Leu Leu Leu 100 105 110 Leu Ala Ser Ala Met Val Glu Ala Gly Ala Gly Thr Gly Trp Thr Val 115 120 125 Tyr Pro Pro Leu Ala Gly Asn Tyr Ser His Pro Gly Ala Ser Val Asp 130 135 140 Leu Thr Ile Phe Ser Leu His Leu Ala Gly Val Ser Ser Ile Leu Gly 145 150 155 160 Ala Ile Asn Phe Ile Thr Thr Ile Ile Asn Met Lys Pro Pro Ala Met 165 170 175 Thr Gln Tyr Gln Thr Pro Leu Phe Val Trp Ser Val Leu Ile Thr Ala 180 185 190 Val Leu Leu Leu Leu Ser Leu Pro Val Leu Ala Ala Gly Ile Thr Met 195 200 205 Leu Leu Thr Asp Arg Asn Leu Asn Thr Thr Phe Phe Asp Pro Ala Gly 210 215 220 Gly Gly Asp Pro Ile Leu Tyr Gln His Leu Phe Trp Phe Phe Gly His 225 230 235 240 Pro Glu Val Tyr Ile Leu Ile Leu Pro Gly Phe Gly Met Ile Ser His 245 250 255 Ile Val Thr Tyr Tyr Ser Gly Lys Lys Glu Pro Phe Gly Tyr Met Gly 260 265 270 Met Val Trp Ala Met Met Ser Ile Gly Phe Leu Gly Phe Ile Val Trp 275 280 285 Ala His His Met Phe Thr Val Gly Met Asp Val Asp Thr Arg Ala Tyr 290 295 300 Phe Thr Ser Ala Thr Met Ile Ile Ala Ile Pro Thr Gly Val Lys Val 305 310 315 320 Phe Ser Trp Leu Ala Thr Leu His Gly Ser Asn Met Lys Trp Ser Ala 325 330 335 Ala Val Leu Trp Ala Leu Gly Phe Ile Phe Leu Phe Thr Val Gly Gly 340 345 350 Leu Thr Gly Ile Val Leu Ala Asn Ser Ser Leu Asp Ile Val Leu His 355 360 365 Asp Thr Tyr Tyr Val Val Ala His Phe His Tyr Val Leu Ser Met Gly 370 375 380 Ala Val Phe Ala Ile Met Gly Gly Phe Ile His Trp Phe Pro Leu Phe 385 390 395 400 Ser Gly Tyr Thr Leu Asp Gln Thr Tyr Ala Lys Ile His Phe Thr Ile 405 410 415 Met Phe Ile Gly Val Asn Leu Thr Phe Phe Pro Gln His Phe Leu Gly 420 425 430 Leu Ser Gly Met Pro Arg Arg Tyr Ser Asp Tyr Pro Asp Ala Tyr Thr 435 440 445 Thr Trp Asn Ile Leu Ser Ser Val Gly Ser Phe Ile Ser Leu Thr Ala 450 455 460 Val Met Leu Met Ile Phe Met Ile Trp Glu Ala Phe Ala Ser Lys Arg 465 470 475 480 Lys Val Leu Met Val Glu Glu Pro Ser Met Asn Leu Glu Trp Leu Tyr 485 490 495 Gly Cys Pro Pro Pro Tyr His Thr Phe Glu Glu Pro Val Tyr Met Lys 500 505 510 Ser 227 amino acids amino acid linear 16 Met Ala His Ala Ala Gln Val Gly Leu Gln Asp Ala Thr Ser Pro Ile 1 5 10 15 Met Glu Glu Leu Ile Thr Phe His Asp His Ala Leu Met Ile Ile Phe 20 25 30 Leu Ile Cys Phe Leu Val Leu Tyr Ala Leu Phe Leu Thr Leu Thr Thr 35 40 45 Lys Leu Thr Asn Thr Asn Ile Ser Asp Ala Gln Glu Met Glu Thr Val 50 55 60 Trp Thr Ile Leu Pro Ala Ile Ile Leu Val Leu Ile Ala Leu Pro Ser 65 70 75 80 Leu Arg Ile Leu Tyr Met Thr Asp Glu Val Asn Asp Pro Ser Leu Thr 85 90 95 Ile Lys Ser Ile Gly His Gln Trp Tyr Trp Thr Tyr Glu Tyr Thr Asp 100 105 110 Tyr Gly Gly Leu Ile Phe Asn Ser Tyr Met Leu Pro Pro Leu Phe Leu 115 120 125 Glu Pro Gly Asp Leu Arg Leu Leu Asp Val Asp Asn Arg Val Val Leu 130 135 140 Pro Ile Glu Ala Pro Ile Arg Met Met Ile Thr Ser Gln Asp Val Leu 145 150 155 160 His Ser Trp Ala Val Pro Thr Leu Gly Leu Lys Thr Asp Ala Ile Pro 165 170 175 Gly Arg Leu Asn Gln Thr Thr Phe Thr Ala Thr Arg Pro Gly Val Tyr 180 185 190 Tyr Gly Gln Cys Ser Glu Ile Cys Gly Ala Asn His Ser Phe Met Pro 195 200 205 Ile Val Leu Glu Leu Ile Pro Leu Lys Ile Phe Glu Met Gly Pro Val 210 215 220 Phe Thr Leu 225 68 amino acids amino acid linear 17 Met Pro Gln Leu Asn Thr Thr Val Trp Pro Thr Met Ile Thr Pro Met 1 5 10 15 Leu Leu Thr Leu Phe Leu Ile Thr Gln Leu Lys Met Leu Asn Thr Asn 20 25 30 Tyr His Leu Pro Pro Ser Pro Lys Pro Met Lys Met Lys Asn Tyr Asn 35 40 45 Lys Pro Trp Glu Pro Lys Trp Thr Lys Ile Cys Ser Leu His Ser Leu 50 55 60 Pro Pro Gln Ser 65 226 amino acids amino acid linear 18 Met Asn Glu Asn Leu Phe Ala Ser Phe Ile Ala Pro Thr Ile Leu Gly 1 5 10 15 Leu Pro Ala Ala Val Leu Ile Ile Leu Phe Pro Pro Leu Leu Ile Pro 20 25 30 Thr Ser Lys Tyr Leu Ile Asn Asn Arg Leu Ile Thr Thr Gln Gln Trp 35 40 45 Leu Ile Lys Leu Thr Ser Lys Gln Met Met Thr Met His Asn Thr Lys 50 55 60 Gly Arg Thr Trp Ser Leu Met Leu Val Ser Leu Ile Ile Phe Ile Ala 65 70 75 80 Thr Thr Asn Leu Leu Gly Leu Leu Pro His Ser Phe Thr Pro Thr Thr 85 90 95 Gln Leu Ser Met Asn Leu Ala Met Ala Ile Pro Leu Trp Ala Gly Thr 100 105 110 Val Ile Met Gly Phe Arg Ser Lys Ile Lys Asn Ala Leu Ala His Phe 115 120 125 Leu Pro Gln Gly Thr Pro Thr Pro Leu Ile Pro Met Leu Val Ile Ile 130 135 140 Glu Thr Ile Ser Leu Leu Ile Gln Pro Met Ala Leu Ala Val Arg Leu 145 150 155 160 Thr Ala Asn Ile Thr Ala Gly His Leu Leu Met His Leu Ile Gly Ser 165 170 175 Ala Thr Leu Ala Met Ser Thr Ile Asn Leu Pro Ser Thr Leu Ile Ile 180 185 190 Phe Thr Ile Leu Ile Leu Leu Thr Ile Leu Glu Ile Ala Val Ala Leu 195 200 205 Ile Gln Ala Tyr Val Phe Thr Leu Leu Val Ser Leu Tyr Leu His Asp 210 215 220 Asn Thr 225 261 amino acids amino acid linear 19 Met Thr His Gln Ser His Ala Tyr His Met Val Lys Pro Ser Pro Trp 1 5 10 15 Pro Leu Thr Gly Ala Leu Ser Ala Leu Leu Met Thr Ser Gly Leu Ala 20 25 30 Met Trp Phe His Phe His Ser Met Thr Leu Leu Met Leu Gly Leu Leu 35 40 45 Thr Asn Thr Leu Thr Met Tyr Gln Trp Trp Arg Asp Val Thr Arg Glu 50 55 60 Ser Thr Tyr Gln Gly His His Thr Pro Pro Val Gln Lys Gly Leu Arg 65 70 75 80 Tyr Gly Met Ile Leu Phe Ile Thr Ser Glu Val Phe Phe Phe Ala Gly 85 90 95 Phe Phe Trp Ala Phe Tyr His Ser Ser Leu Ala Pro Thr Pro Gln Leu 100 105 110 Gly Gly His Trp Pro Pro Thr Gly Ile Thr Pro Leu Asn Pro Leu Glu 115 120 125 Val Pro Leu Leu Asn Thr Ser Val Leu Leu Ala Ser Gly Val Ser Ile 130 135 140 Thr Trp Ala His His Ser Leu Met Glu Asn Asn Arg Asn Gln Met Ile 145 150 155 160 Gln Ala Leu Leu Ile Thr Ile Leu Leu Gly Leu Tyr Phe Thr Leu Leu 165 170 175 Gln Ala Ser Glu Tyr Phe Glu Ser Pro Phe Thr Ile Ser Asp Gly Ile 180 185 190 Tyr Gly Ser Thr Phe Phe Val Ala Thr Gly Phe His Gly Leu His Val 195 200 205 Ile Ile Gly Ser Thr Phe Leu Thr Ile Cys Phe Ile Arg Gln Leu Met 210 215 220 Phe His Phe Thr Ser Lys His His Phe Gly Phe Glu Ala Ala Ala Trp 225 230 235 240 Tyr Trp His Phe Val Asp Val Val Trp Leu Phe Leu Tyr Val Ser Ile 245 250 255 Tyr Trp Trp Gly Ser 260 115 amino acids amino acid linear 20 Met Asn Phe Ala Leu Ile Leu Met Ile Asn Thr Leu Leu Ala Leu Leu 1 5 10 15 Leu Met Ile Ile Thr Phe Trp Leu Pro Gln Leu Asn Gly Tyr Met Glu 20 25 30 Lys Ser Thr Pro Tyr Glu Cys Gly Phe Asp Pro Met Ser Pro Ala Arg 35 40 45 Val Pro Phe Ser Met Lys Phe Phe Leu Val Ala Ile Thr Phe Leu Leu 50 55 60 Phe Asp Leu Glu Ile Ala Leu Leu Leu Pro Leu Pro Trp Ala Leu Gln 65 70 75 80 Thr Thr Asn Leu Pro Leu Met Val Met Ser Ser Leu Leu Leu Ile Ile 85 90 95 Ile Leu Ala Leu Ser Leu Ala Tyr Glu Trp Leu Gln Lys Gly Leu Asp 100 105 110 Trp Thr Glu 115 98 amino acids amino acid linear 21 Met Pro Leu Ile Tyr Met Asn Ile Met Leu Ala Phe Thr Ile Ser Leu 1 5 10 15 Leu Gly Met Leu Val Tyr Arg Ser His Leu Met Ser Ser Leu Leu Cys 20 25 30 Leu Glu Gly Met Met Leu Ser Leu Phe Ile Met Ala Thr Leu Met Thr 35 40 45 Leu Asn Thr His Ser Leu Leu Ala Asn Ile Val Pro Ile Ala Met Leu 50 55 60 Val Phe Ala Ala Cys Glu Ala Ala Val Gly Leu Ala Leu Leu Val Ser 65 70 75 80 Ile Ser Asn Thr Tyr Gly Leu Asp Tyr Val His Asn Leu Asn Leu Leu 85 90 95 Gln Cys 459 amino acids amino acid linear 22 Met Leu Lys Leu Ile Val Pro Thr Ile Met Leu Leu Pro Leu Thr Trp 1 5 10 15 Leu Ser Lys Lys His Met Ile Trp Ile Asn Thr Thr Thr His Ser Leu 20 25 30 Ile Ile Ser Ile Ile Pro Leu Leu Phe Phe Asn Gln Ile Asn Asn Asn 35 40 45 Leu Phe Ser Cys Ser Pro Thr Phe Ser Ser Asp Pro Leu Thr Thr Pro 50 55 60 Leu Leu Met Leu Thr Thr Trp Leu Leu Pro Leu Thr Ile Met Ala Ser 65 70 75 80 Gln Arg His Leu Ser Ser Glu Pro Leu Ser Arg Lys Lys Leu Tyr Leu 85 90 95 Ser Met Leu Ile Ser Leu Gln Ile Ser Leu Ile Met Thr Phe Thr Ala 100 105 110 Thr Glu Leu Ile Met Phe Tyr Ile Phe Phe Glu Thr Thr Leu Ile Pro 115 120 125 Thr Leu Ala Ile Ile Thr Arg Trp Gly Asn Gln Pro Glu Arg Leu Asn 130 135 140 Ala Gly Thr Tyr Phe Leu Phe Tyr Thr Leu Val Gly Ser Leu Pro Leu 145 150 155 160 Leu Ile Ala Leu Ile Tyr Thr His Asn Thr Leu Gly Ser Leu Asn Ile 165 170 175 Leu Leu Leu Thr Leu Thr Ala Gln Glu Leu Ser Asn Ser Trp Ala Asn 180 185 190 Asn Leu Met Trp Leu Ala Tyr Thr Met Ala Phe Met Val Lys Met Pro 195 200 205 Leu Tyr Gly Leu His Leu Trp Leu Pro Lys Ala His Val Glu Ala Pro 210 215 220 Ile Ala Gly Ser Met Val Leu Ala Ala Val Leu Leu Lys Leu Gly Gly 225 230 235 240 Tyr Gly Met Met Arg Leu Thr Leu Ile Leu Asn Pro Leu Thr Lys His 245 250 255 Met Ala Tyr Pro Phe Leu Val Leu Ser Leu Trp Gly Met Ile Met Thr 260 265 270 Ser Ser Ile Cys Leu Arg Gln Thr Asp Leu Lys Ser Leu Ile Ala Tyr 275 280 285 Ser Ser Ile Ser His Met Ala Leu Val Val Thr Ala Ile Leu Ile Gln 290 295 300 Thr Pro Trp Ser Phe Thr Gly Ala Val Ile Leu Met Ile Ala His Gly 305 310 315 320 Leu Thr Ser Ser Leu Leu Phe Cys Leu Ala Asn Ser Asn Tyr Glu Arg 325 330 335 Thr His Ser Arg Ile Met Ile Leu Ser Gln Gly Leu Gln Thr Leu Leu 340 345 350 Pro Leu Met Ala Phe Trp Trp Leu Leu Ala Ser Leu Ala Asn Leu Ala 355 360 365 Leu Pro Pro Thr Ile Asn Leu Leu Gly Glu Leu Ser Val Leu Val Thr 370 375 380 Thr Phe Ser Trp Ser Asn Ile Thr Leu Leu Leu Thr Gly Leu Asn Met 385 390 395 400 Leu Val Thr Ala Leu Tyr Ser Leu Tyr Met Phe Thr Thr Thr Gln Trp 405 410 415 Gly Ser Leu Thr His His Ile Asn Asn Met Lys Pro Ser Phe Thr Arg 420 425 430 Glu Asn Thr Leu Met Phe Met His Leu Ser Pro Ile Leu Leu Leu Ser 435 440 445 Leu Asn Pro Asp Ile Ile Thr Gly Phe Ser Ser 450 455 603 amino acids amino acid linear 23 Met Thr Met His Thr Thr Met Thr Thr Leu Thr Leu Thr Ser Leu Ile 1 5 10 15 Pro Pro Ile Leu Thr Thr Leu Val Asn Pro Asn Lys Lys Asn Ser Tyr 20 25 30 Pro His Tyr Val Lys Ser Ile Val Ala Ser Thr Phe Ile Ile Ser Leu 35 40 45 Phe Pro Thr Thr Met Phe Met Cys Leu Asp Gln Glu Val Ile Ile Ser 50 55 60 Asn Trp His Trp Ala Thr Thr Gln Thr Thr Gln Leu Ser Leu Ser Phe 65 70 75 80 Lys Leu Asp Tyr Phe Ser Met Met Phe Ile Pro Val Ala Leu Phe Val 85 90 95 Thr Trp Ser Ile Met Glu Phe Ser Leu Trp Tyr Met Asn Ser Asp Pro 100 105 110 Asn Ile Asn Gln Phe Phe Lys Tyr Leu Leu Ile Phe Leu Ile Thr Met 115 120 125 Leu Ile Leu Val Thr Ala Asn Asn Leu Phe Gln Leu Phe Ile Gly Trp 130 135 140 Glu Gly Val Gly Ile Met Ser Phe Leu Leu Ile Ser Trp Trp Tyr Ala 145 150 155 160 Arg Ala Asp Ala Asn Thr Ala Ala Ile Gln Ala Ile Leu Tyr Asn Arg 165 170 175 Ile Gly Asp Ile Gly Phe Ile Leu Ala Leu Ala Trp Phe Ile Leu His 180 185 190 Ser Asn Ser Trp Asp Pro Gln Gln Met Ala Leu Leu Asn Ala Asn Pro 195 200 205 Ser Leu Thr Pro Leu Leu Gly Leu Leu Leu Ala Ala Ala Gly Lys Ser 210 215 220 Ala Gln Leu Gly Leu His Pro Trp Leu Pro Ser Ala Met Glu Gly Pro 225 230 235 240 Thr Pro Val Ser Ala Leu Leu His Ser Ser Thr Met Val Val Ala Gly 245 250 255 Ile Phe Leu Leu Ile Arg Phe His Pro Leu Ala Glu Asn Ser Pro Leu 260 265 270 Ile Gln Thr Leu Thr Leu Cys Leu Gly Ala Ile Thr Thr Leu Phe Ala 275 280 285 Ala Val Cys Ala Leu Thr Gln Asn Asp Ile Lys Lys Ile Val Ala Phe 290 295 300 Ser Thr Ser Ser Gln Leu Gly Leu Met Met Val Thr Ile Gly Ile Asn 305 310 315 320 Gln Pro His Leu Ala Phe Leu His Ile Cys Thr His Ala Phe Phe Lys 325 330 335 Ala Met Leu Phe Met Cys Ser Gly Ser Ile Ile His Asn Leu Asn Asn 340 345 350 Glu Gln Asp Ile Arg Lys Met Gly Gly Leu Leu Lys Thr Met Pro Leu 355 360 365 Thr Ser Thr Ser Leu Thr Ile Gly Ser Leu Ala Leu Ala Gly Met Pro 370 375 380 Phe Leu Thr Gly Phe Tyr Ser Lys Asp His Ile Ile Glu Thr Ala Asn 385 390 395 400 Met Ser Tyr Thr Asn Ala Trp Ala Leu Ser Ile Thr Leu Ile Ala Thr 405 410 415 Ser Leu Thr Ser Ala Tyr Ser Thr Arg Met Ile Leu Leu Thr Leu Thr 420 425 430 Gly Gln Pro Arg Phe Pro Thr Leu Thr Asn Ile Asn Glu Asn Asn Pro 435 440 445 Thr Leu Leu Asn Pro Ile Lys Arg Leu Ala Ala Gly Ser Leu Phe Ala 450 455 460 Gly Phe Leu Ile Thr Asn Asn Ile Ser Pro Ala Ser Pro Phe Gln Thr 465 470 475 480 Thr Ile Pro Leu Tyr Leu Lys Leu Thr Ala Leu Ala Val Thr Phe Leu 485 490 495 Gly Leu Leu Thr Ala Leu Asp Leu Asn Tyr Leu Thr Asn Lys Leu Lys 500 505 510 Met Lys Ser Pro Leu Cys Thr Phe Tyr Phe Ser Asn Met Leu Gly Phe 515 520 525 Tyr Pro Ser Ile Thr His Arg Thr Ile Pro Tyr Leu Gly Leu Leu Thr 530 535 540 Ser Gln Asn Leu Pro Leu Leu Leu Leu Asp Leu Thr Trp Leu Glu Lys 545 550 555 560 Leu Leu Pro Lys Thr Ile Ser Gln His Gln Ile Ser Thr Ser Ile Ile 565 570 575 Thr Ser Thr Gln Lys Gly Met Ile Lys Leu Tyr Phe Leu Ser Phe Phe 580 585 590 Phe Pro Leu Ile Leu Thr Leu Leu Leu Ile Thr 595 600 174 amino acids amino acid linear 24 Met Met Tyr Ala Leu Phe Leu Leu Ser Val Gly Leu Val Met Gly Phe 1 5 10 15 Val Gly Phe Ser Ser Lys Pro Ser Pro Ile Tyr Gly Gly Leu Val Leu 20 25 30 Ile Val Ser Gly Val Val Gly Cys Val Ile Ile Leu Asn Phe Gly Gly 35 40 45 Gly Tyr Met Gly Leu Met Val Phe Leu Ile Tyr Leu Gly Gly Met Met 50 55 60 Val Val Phe Gly Tyr Thr Thr Ala Met Ala Ile Glu Glu Tyr Pro Glu 65 70 75 80 Ala Trp Gly Ser Gly Val Glu Val Leu Val Ser Val Leu Val Gly Leu 85 90 95 Ala Met Glu Val Gly Leu Val Leu Trp Val Lys Glu Tyr Asp Gly Val 100 105 110 Val Val Val Val Asn Phe Asn Ser Val Gly Ser Trp Met Ile Tyr Glu 115 120 125 Gly Glu Gly Ser Gly Leu Ile Arg Glu Asp Pro Ile Gly Ala Gly Ala 130 135 140 Leu Tyr Asp Tyr Gly Arg Trp Leu Val Val Val Thr Gly Trp Thr Leu 145 150 155 160 Phe Val Gly Val Tyr Ile Val Ile Glu Ile Ala Arg Gly Asn 165 170 380 amino acids amino acid linear 25 Met Thr Pro Met Arg Lys Ile Asn Pro Leu Met Lys Leu Ile Asn His 1 5 10 15 Ser Phe Ile Asp Leu Pro Thr Pro Ser Asn Ile Ser Ala Trp Trp Asn 20 25 30 Phe Gly Ser Leu Leu Gly Ala Cys Leu Ile Leu Gln Ile Thr Thr Gly 35 40 45 Leu Phe Leu Ala Met His Tyr Ser Pro Asp Ala Ser Thr Ala Phe Ser 50 55 60 Ser Ile Ala His Ile Thr Arg Asp Val Asn Tyr Gly Trp Ile Ile Arg 65 70 75 80 Tyr Leu His Ala Asn Gly Ala Ser Met Phe Phe Ile Cys Leu Phe Leu 85 90 95 His Ile Gly Arg Gly Leu Tyr Tyr Gly Ser Phe Leu Tyr Ser Glu Thr 100 105 110 Trp Asn Ile Gly Ile Ile Leu Leu Leu Ala Thr Met Ala Thr Ala Phe 115 120 125 Met Gly Tyr Val Leu Pro Trp Gly Gln Met Ser Phe Trp Gly Ala Thr 130 135 140 Val Ile Thr Asn Leu Leu Ser Ala Ile Pro Tyr Ile Gly Thr Asp Leu 145 150 155 160 Val Gln Trp Ile Trp Gly Gly Tyr Ser Val Asp Ser Pro Thr Leu Thr 165 170 175 Arg Phe Phe Thr Phe His Phe Ile Leu Pro Phe Ile Ile Ala Ala Leu 180 185 190 Ala Thr Leu His Leu Leu Phe Leu His Glu Thr Gly Ser Asn Asn Pro 195 200 205 Leu Gly Ile Thr Ser His Ser Asp Lys Ile Thr Phe His Pro Tyr Tyr 210 215 220 Thr Ile Lys Asp Ala Leu Gly Leu Leu Leu Phe Leu Leu Ser Leu Met 225 230 235 240 Thr Leu Thr Leu Phe Ser Pro Asp Leu Leu Gly Asp Pro Asp Asn Tyr 245 250 255 Thr Leu Ala Asn Pro Leu Asn Thr Pro Pro His Ile Lys Pro Glu Trp 260 265 270 Tyr Phe Leu Phe Ala Tyr Thr Ile Leu Arg Ser Val Pro Asn Lys Leu 275 280 285 Gly Gly Val Leu Ala Leu Leu Leu Ser Ile Leu Ile Leu Ala Met Ile 290 295 300 Pro Ile Leu His Met Ser Lys Gln Gln Ser Met Met Phe Arg Pro Leu 305 310 315 320 Ser Gln Ser Leu Tyr Trp Leu Leu Ala Ala Asp Leu Leu Ile Leu Thr 325 330 335 Trp Ile Gly Gly Gln Pro Val Ser Tyr Pro Phe Thr Ile Ile Gly Gln 340 345 350 Val Ala Ser Val Leu Tyr Phe Thr Thr Ile Leu Ile Leu Met Pro Thr 355 360 365 Ile Ser Leu Ile Glu Asn Lys Met Leu Lys Trp Ala 370 375 380 823 base pairs nucleic acid single linear 26 CCCACATTAG GCTTAAAAAC AGATGCAATT CCCGGACGTC TAAACCAAAC CACTTTCACC 60 GCTACACGAC CGGGGGTATA CTACGGTCAA TGCTCTGAAA TCTGTGGAGC AAACCACAGT 120 TTCATGCCCA TCGTCCTAGA ATTAATTCCC CTAAAAATCT TTGAAATAGG GCCCGTATTT 180 ACCCTATAGC ACCCCCTCTA CCCCCTCTAG AGCCCACTGT AAAGCTAACT TAGCATTAAC 240 CTTTTAAGTT AAAGATTAAG AGAACCAACA CCTCTTTACA GTGAAATGCC CCAACTAAAT 300 ACTACCGTAT GGCCCACCAT AATTACCCCC ATACTCCTTA CACTATTCCT CATCACCCAA 360 CTAAAAATAT TAAACACAAA CTACCACCTA CCTCCCTCAC CAAAGCCCAT AAAAATAAAA 420 AATTATAACA AACCCTGAGA ACCAAAATGA ACGAAAATCT GTTCGCTTCA TTCATTGCCC 480 CCACAATCCT AGGCCTACCC GCCGCAGTAC TGATCATTCT ATTTCCCCCT CTATTGATCC 540 CCACCTCCAA ATATCTCATC AACAACCGAC TAATCACCAC CCAACAATGA CTAATCAAAC 600 TAACCTCAAA ACAAATGATA ACCATACACA ACACTAAAGG ACGAACCTGA TCTCTTATAC 660 TAGTATCCTT AATCATTTTT ATTGCCACAA CTAACCTCCT CGGACTCCTG CCTCACTCAT 720 TTACACCAAC CACCCAACTA TCTATAAACC TAGCCATGGC CATCCCCTTA TGAGCGGGCA 780 CAGTGATTAT AGGCTTTCGC TCTAAGATTA AAAATGCCCT AGC 823 

What is claimed is:
 1. A method for determining the risk for or presence of a disease associated with altered mitochondrial function in a first subject suspected of having or being at risk for having such a disease, comprising: quantifying, in each of a first and a second biological sample containing extramitochondrial DNA and mitochondrial DNA, (i) an amount y of a mitochondrial DNA sequence, and (ii) an amount x of an extramitochondrial DNA sequence that corresponds to the mitochondrial DNA sequence, said first biological sample being obtained from a first subject suspected of having or being at risk for having a disease associated with altered mitochondrial function and said second sample being obtained from a second subject known to be free of a risk or presence of a disease associated with altered mitochondrial function; and comparing a ratio r for each of the first and second biological samples using the formula: r=x/(x+y) and therefrom determining the risk or presence of the disease.
 2. The method of claim 1 wherein the ratio r is calculated by a method comprising: contacting a biological sample containing extramitochondrial DNA and mitochondrial DNA with an oligonucleotide primer having a nucleotide sequence that is complementary to a sequence present in said extramitochondrial DNA and present in said mitochondrial DNA, under conditions and for a time sufficient to allow hybridization of said primer to the extramitochondrial DNA and to the mitochondrial DNA; and detecting hybridization of the primer to the extramitochondrial DNA and to the mitochondrial DNA, and therefrom quantifying the extramitochondrial DNA and the mitochondrial DNA.
 3. The method of claim 1 wherein the ratio r is calculated by a method comprising: contacting a sample containing amplified extramitochondrial DNA and mitochondrial DNA with an oligonucleotide primer having a nucleotide sequence that is complementary to a sequence present in said amplified extramitochondrial DNA and present in said amplified mitochondrial DNA, under conditions and for a time sufficient to allow hybridization of said primer to the extramitochondrial DNA and to the mitochondrial DNA; and detecting hybridization of the primer to the extramitochondrial DNA and to the mitochondrial DNA, and therefrom quantifying the extramitochondrial DNA and the mitochondrial DNA.
 4. The method of claim 1 wherein the ratio r is calculated by a method comprising: contacting a biological sample containing extramitochondrial DNA and mitochondrial DNA with an oligonucleotide primer having a nucleotide sequence that is complementary to a sequence present in said extramitochondrial DNA and present in said mitochondrial DNA, under conditions and for a time sufficient to allow hybridization of said primer to the extramitochondrial DNA and to the mitochondrial DNA; and detecting hybridization and extension of the primer to the extramitochondrial DNA to produce a first product and hybridization and extension of the primer to the mitochondrial DNA to produce a second product distinguishable from said first product, and therefrom quantifying the extramitochondrial DNA and the mitochondrial DNA.
 5. The method of claim 1 wherein the ratio r is calculated by a method comprising: contacting a sample containing amplified extramitochondrial DNA and mitochondrial DNA with an oligonucleotide primer having a nucleotide sequence that is complementary to a sequence present in said amplified extramitochondrial DNA and present in said amplified mitochondrial DNA, under conditions and for a time sufficient to allow hybridization of said primer to the extramitochondrial DNA and to the mitochondrial DNA; and detecting hybridization and extension of the primer to the extramitochondrial DNA to produce a first product and hybridization and extension of the primer to the mitochondrial DNA to produce a second product distinguishable from said first product, and therefrom quantifying the extramitochondrial DNA and the mitochondrial DNA.
 6. The method of claim 1 wherein the biological sample is treated by heating said sample in water to lyse cells contained in the sample, and then extracting cellular DNA from said lysed cells using an aqueous DNA extraction procedure.
 7. The method of claim 6 wherein the sample comprises a crude buffy coat fraction of whole blood.
 8. The method of claim 1, further comprising the step of determining the ApoE genotype of the first subject and correlating said genotype with the risk or presence of disease.
 9. The method of claim 1 wherein the disease associated with altered mitochondrial function is selected from the group consisting of Alzheimer's Disease, Huntington's Disease, Parkinson's Disease, dystonia, schizophrenia, non-insulin dependent diabetes mellitus, mitochondrial encephalopathy, lactic acidosis, and stroke, myoclonic epilepsy ragged red fiber syndrome, and Leber's hereditary optic neuropathy.
 10. The method of claim 1 wherein the ratio r for the biological sample is calculated by determining the presence in said sample of a nucleotide sequence selected from the group consisting of SEQ ID NO:1, a portion of SEQ ID NO:1, SEQ ID NO:3 and a portion of SEQ ID NO:3.
 11. The method of claim 10 wherein the portion of the nucleotide sequence of SEQ ID NO:1 corresponds to a portion of the nucleotide sequence of SEQ ID NO:2 encoding a mitochondrial cytochrome c oxidase.
 12. The method of claim 10 wherein the portion of SEQ ID NO:1 corresponds to a portion of a mitochondrial cytochrome c oxidase encoding sequence selected from the group consisting of a portion of a CO1 encoding sequence and a portion of a CO2 encoding sequence.
 13. The method of claim 10 wherein the portion of the nucleotide sequence of SEQ ID NO:1 corresponds to a portion of the nucleotide sequence of SEQ ID NO:2 encoding a mitochondrial ATP synthetase subunit.
 14. The method of claim 10 wherein the portion of SEQ ID NO:1 corresponds to a portion of a mitochondrial ATP synthetase subunit encoding sequence selected from the group consisting of a portion of a sequence encoding ATP synthetase subunit 6 and a portion of a sequence encoding ATP synthetase subunit
 8. 15. The method of claim 10 wherein the nucleotide sequence of SEQ ID NO:1 corresponds to a portion of SEQ ID NO:2 selected from the group consisting of a portion of a sequence encoding ND1, a sequence encoding a portion of ND2 and a sequence encoding a portion of CO3.
 16. The method of claim 10 wherein the portion of the nucleotide sequence of SEQ ID NO:3 corresponds to a portion of the nucleotide sequence of SEQ ID NO:2 encoding a mitochondrial ATP synthetase subunit.
 17. The method of claim 10 wherein the portion of SEQ ID NO:3 corresponds to a portion of a mitochondrial ATP synthetase subunit encoding sequence selected from the group consisting of a portion of a sequence encoding ATP synthetase subunit 6 and a portion of a sequence encoding ATP synthetase subunit
 8. 18. The method of claim 10 wherein the portion of the nucleotide sequence of SEQ ID NO:1 corresponds to a portion of the nucleotide sequence of SEQ ID NO:2 encoding a mitochondrial tRNA.
 19. The method of claim 10 wherein the portion of the nucleotide sequence of SEQ ID NO:3 corresponds to a portion of the nucleotide sequence of SEQ ID NO:2 encoding a mitochondrial tRNA.
 20. A method for determining the risk or presence of a disease associated with altered mitochondrial function in a first subject suspected of having or being at risk for having such a disease, comprising: quantifying, in a first biological sample containing extramitochondrial DNA and mitochondrial DNA, wherein at least one of said extramitochondrial DNA and said mitochondrial DNA is from a first subject suspected of having or being at risk for having a disease associated with altered mitochondrial function, (i) an amount of a first mitochondrial DNA sequence and (ii) an amount of a first extramitochondrial DNA sequence that corresponds to the first mitochondrial DNA sequence; and comparing said amounts of the first mitochondrial DNA sequence and the first extramitochondrial DNA sequence that corresponds to the first mitochondrial DNA sequence to amounts, in a second biological sample from a second subject known to be free of a risk or presence of a disease associated with altered mitochondrial function, of a second mitochondrial DNA sequence and a second extramitochondrial DNA sequence that corresponds to the second mitochondrial DNA sequence, and therefrom determining the risk or presence of the disease. 